TruSeq Ribo Profile (Mammalian) Kit
User Guide
Kit Contents
Consumables and Equipment
TruSeq Ribo Profile (Mammalian) Kit Protocol
Preparation and Isolation of RPFs
Purification of RPFs
Library Preparation
Appendices
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15066016 Rev A
December 2014
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Kit Contents
Table 1 TruSeq Ribo Profile (Mammalian) Kit Components
Component Name
Catalog No.
TruSeq Ribo Profile (Mammalian) Kit ASLPA1212
5X Mammalian Polysome Buffer
ASBHMR1212
TruSeq Ribo Profile Index PCR
SMIP2124
Primers (1-12)
Filter Tubes
ASFT1212
Quantity
1 kit
1 bottle
1 kit
Storage
-20°C
-20°C
-20°C
1 pack
RT
Consumables and Equipment
Consumables and Equipment
RPF Preparation, Isolation, and Purification
Table 2 User-Supplied Consumables
Consumable
*
Supplier
Cycloheximide (50 mg/ml in absolute ethanol)
General lab supplier*
SUPERase•In RNase Inhibitor
Life Technologies, Catalog No.
AM2696
Phosphate-Buffered Saline (PBS)
General lab supplier
Liquid nitrogen
General lab supplier
Isopropyl alcohol (100%)
General lab supplier
Freshly prepared 80% (v/v) ethanol, ice-cold
General lab supplier
illustra MicroSpin S-400 HR Columns
GE Healthcare, Catalog No. 275140-01
Optional: Sucrose
General lab supplier
Optional: NP-40 (10%)
Thermo Fisher, Catalog No. 85124
Optional: Sterile 22–25-gauge needle
General lab supplier
Optional: 10% polyacrylamide/7–8 M urea/TBE gel
General lab supplier
Optional: Wide-bore pipet tips (eg, Pure 200G
sterile tip)
Molecular Bioproducts, Catalog No.
3531
Cycloheximide is available from many vendors. Illumina recommends preparing 15 ml of a 50
mg/ml stock solution using ethanol as the diluent. The compound is light-sensitive and can be
stored up to 1 year at –20°C. Cycloheximide is toxic and proper personal protective equipment
should be used. Refer to the manufacturer's safety data sheet for more information.
Table 3 User-Supplied Equipment
Equipment
Supplier
NanoDrop UV-Vis Spectrophotometer
Thermo Fisher
Microcentrifuge for 0.5 ml and 1.5 ml tubes
General lab supplier
Benchtop centrifuge with fixed-angle rotor
General lab supplier
Library Preparation
Table 4 User-Supplied Consumables
Consumable
Supplier
Ribo-Zero Magnetic Gold Kit
(Human/Mouse/Rat)
Illumina, Catalog No. MRZG12324
RNA Clean & Concentrator-5 Kit
Zymo Research, Catalog No. R1015
TruSeq Ribo Profile (Mammalian) Kit
4
Consumable
Supplier
RNA Clean & Concentrator-25 Kit
Zymo Research, Catalog No. R1017
Denaturing Gel Loading Dye
Ambion, Catalog No. 8546G
6X native gel loading dye containing
Bromophenol Blue
General lab supplier
20/100 Oligo Ladder (1 ng/μl)
IDT, Catalog No. 51-05-15-02
20 bp Ladder
Bayou Biolabs, Catalog No. L-100
12% or 15% polyacrylamide/7–8 M urea/TBE gel
General lab supplier
8% native polyacrylamide/TBE gel
General lab supplier
SYBR Gold
Life Technologies, Catalog No. S11494
Phusion Hi-Fi PCR Master Mix
NEB, Catalog No. M0531
AMPure XP System
Beckman-Coulter Genomics,
Catalog No. A63880
Sterile 20-gauge needle
General lab supplier
Sterile 0.5 ml and 1.5 ml microcentrifuge tubes
General lab supplier
Table 5 User-Supplied Equipment
Equipment
5
Supplier
1.5 ml microcentrifuge
General lab supplier
Shaker-incubator
General lab supplier
Dark-field transilluminator
General lab supplier
Part # 15066016 Rev A
TruSeq Ribo Profile (Mammalian) Kit Protocol
TruSeq Ribo Profile (Mammalian) Kit Protocol
Figure 1 Overview of the TruSeq Ribo Profile (Mammalian) Kit Workflow
TruSeq Ribo Profile (Mammalian) Kit
6
Figure 2 Schematic Representation of the TruSeq Ribo Profile (Mammalian) Kit Process
7
Part # 15066016 Rev A
Preparation and Isolation of RPFs
Preparation and Isolation of RPFs
Cell Extract Preparation and Ribosome Footprinting
Consumables
Item
Quantity
Storage
Supplied By
Cell culture plates and media
User
Liquid nitrogen
User
PBS
10 ml per reaction
5X Mammalian Polysome
Buffer
200 μl per reaction
-20°C
Illumina
DTT (100 mM)
10 μl per reaction
-20°C
Illumina
10% SDS
10 μl per reaction
-20°C
Illumina
10% Triton X-100
100 μl per reaction
-20°C
Illumina
DNase I (1 U/μl)
10 μl per reaction
-20°C
Illumina
Cycloheximide* (50 mg/ml)
2 μl per reaction
-20°C
Illumina
-20°C
Illumina
Nuclease-Free Water
User
NOTE
Briefly centrifuge tubes to collect reagents at the bottom of the tubes.
*Extra cycloheximide is required when the cells are treated with PBS.
Procedure
1
Prepare 1 ml of Mammalian Lysis Buffer for each sample. Chill the buffer to 4°C.
Reagent
5x Mammalian Polysome Buffer
10% Triton X-100
100 mM DTT
DNase I (1U/μl)
50 mg/ml cycloheximide
10% NP-40 (optional) or
Nuclease-Free Water
Nuclease-Free Water
Total Volume per Sample
Volume (µl)
200
100
10
10
2
10
668
1,000
2
Grow adherent mammalian cells on 15 cm plates in liquid media. The required cell
density will depend on the cell line and growth conditions. A cell density of 5–50
million cells/ml is acceptable (see step 6 in this procedure). Treat the cells with
cycloheximide prior to lysis, aspirate the growth media, supplement the plate with
fresh media containing cycloheximide (final concentration 0.1 mg/ml), and incubate
the cells for 1 minute.
3
Aspirate the media and place the cells on ice. Rinse the cells with 10 ml of ice-cold
PBS supplemented with cycloheximide (final concentration 0.1 mg/ml).
TruSeq Ribo Profile (Mammalian) Kit
8
4
Aspirate and discard the PBS. Add 800 μl of Mammalian Lysis Buffer to the plate.
Scrape the cells extensively and (optionally) draw up and expel through a sterile 22–
25-gauge needle to lyse the cells completely. Transfer the cell lysate to a fresh tube
that has been chilled on ice.
5
Incubate 10 minutes on ice with periodic inversions.
6
Clarify the lysate by centrifugation for 10 minutes at 20,000 × g at 4°C. Transfer the
supernatant to a fresh tube that has been chilled on ice. Expect to recover ~1 ml of
clarified lysate.
7
Prepare a 1:10 dilution of the collected lysate using Nuclease-Free Water. Use a water
blank and a 1:10 dilution of Mammalian Lysis Buffer as a standard. Record an A260
reading using a spectrophotometer. Calculate the A260/ml concentration of the lysate
(see Equation 1). This number will be used to calculate the amount of TruSeq Ribo
Profile Nuclease to generate RPFs in step 1 of Ribosome Footprinting with TruSeq Ribo
Profile Nuclease.
Equation 1: (A260 cell lysate – A260 Mammalian Lysis Buffer) × 10 dilution factor =
A260/ml
8
For each cell lysis, create aliquots: a 100 μl aliquot (for use in the next step) and a
200 μl aliquot (for use in step 1 of Ribosome Footprinting with TruSeq Ribo Profile
Nuclease). Keep on ice.
SAFE STOPPING POINT
Optional: Freeze 100 μl aliquots of the lysate in liquid nitrogen and store at -80°C.
9
To the 100 μl aliquot of cell lysate, add 10 μl of 10% SDS and mix. This material will
be used as Total RNA (nonfootprinted RNA). Place on ice for purification in step 2 of
MicroSpin S-400 Columns or Sucrose Cushion. Alternatively, the sample can be frozen
in liquid nitrogen and stored at -80°C until ready for use.
Ribosome Footprinting with TruSeq Ribo Profile Nuclease
Consumables
Item
Quantity
Storage
Liquid nitrogen
User
TruSeq Ribo Profile Nuclease
(10 U/μl)
SUPERase•In RNase
Inhibitor
Supplied By
15 μl per reaction
-20°C
Illumina
-20°C
User
Procedure
1
9
To the 200 μl aliquot of clarified lysate from step 8 in Cell Extract Preparation and
Ribosome Footprinting, add 5 units of TruSeq Ribo Profile Nuclease for each A260 of
lysate.
For example, 50 A260/ml lysate × 0.2 ml lysate × 5 U/A260 TruSeq Ribo Profile Nuclease
= 50 U of TruSeq Nuclease.
Part # 15066016 Rev A
Optionally, titrate the nuclease digestion as described in Optimizing Nuclease Digestion
to Generate RPFs on page 25.
2
Incubate the reactions at room temperature for 45 minutes with gentle mixing. Freeze
any unused lysate with liquid nitrogen and store at -80°C.
NOTE
During incubation, prepare the MicroSpin S-400 columns as described in MicroSpin S400 Columns on page 11.
3
Stop the reactions by adding 15 μl of SUPERase•In RNase Inhibitor and chill the
samples on ice for use in MicroSpin S-400 Columns on page 11.
TruSeq Ribo Profile (Mammalian) Kit
10
Preparation and Isolation of RPFs
NOTE
An absorbance of 50 A260 /ml is typical with 50 million cells. This value will vary,
depending on cell line.
Purification of RPFs
RPFs can be purified by either MicroSpin S-400 columns (MicroSpin S-400 Columns on
page 11) or sucrose cushion ultracentrifugation (Sucrose Cushion on page 12).
NOTE
Do not purify the samples labeled Total RNA with the Microspin S-400 spin columns. Keep
these samples frozen for use in step 7 of MicroSpin S-400 Columns on page 11 or Sucrose
Cushion on page 12.
MicroSpin S-400 Columns
Consumables
Item
Quantity
Storage
Supplied By
5X Mammalian Polysome
Buffer
600 μl per reaction
-20°C
Illumina
Nuclease-Free Water
2.4 ml per reaction
-20°C
Illumina
illustra MicroSpin S-400 HR
Columns
RNA Clean & Concentrator-25
Kit
User
User
Procedure
1
For each sample, prepare 3 ml of 1X Mammalian Polysome Buffer by combining 600
μl of 5X Buffer with 2.4 ml of Nuclease-Free Water.
2
Invert the MicroSpin S-400 column several times to resuspend the resin. Tap the
column to remove any bubbles that may form in the resin as it settles.
3
Open the column on both ends and allow the buffer to drip out under gravity. If the
column does not dripping freely, use a clean gloved finger to cover the top of the
column. Press down gently to create pressure and initiate flow.
NOTE
Steps 2–3 can be done during nuclease digestion of the RPFs (see Ribosome Footprinting
with TruSeq Ribo Profile Nuclease on page 9). Do not allow the column to dry.
11
4
Attach a collection tube and centrifuge for 4 minutes at 600 × g in a fixed-angle,
benchtop centrifuge at room temperature. Discard the flow-through and transfer the
column to a 1.5 ml tube.
5
Immediately apply 100 μl of nuclease-digested RPF sample (from step 3 in Ribosome
Footprinting with TruSeq Ribo Profile Nuclease on page 9). Keep the remaining ~100 μl
of the sample on ice or freeze in liquid nitrogen and store at -80°C in case it is
needed in step 7. Centrifuge for 2 minutes at 600 × g and collect the flow-through.
Add 10 μl of 10% SDS. This material will be used as RPF RNA.
6
Purify both Total RNA samples and RPF RNA samples using a modified RNA Clean
& Concentrator-25 Kit method:
Part # 15066016 Rev A
Add RNA Binding Buffer and ethanol and mix:
Reagent
RNA Binding
Buffer
100% Ethanol
b
c
Total RNA
Samples
220 μl
RPF
Samples
220 μl
220 μl
495 μl
Continue with purification according to the manufacturer’s instructions.
Elute the samples with 26 μl of Nuclease-Free Water. Expect to recover ~25 μl.
7
Quantify by NanoDrop spectrophotometry. You will need about 1–5 μg of Total RNA
and 1–5 μg of RPF RNA for the Ribo-Zero rRNA removal treatment in Primary rRNA
Depletion on page 13.
a If < 1 μg of Total RNA has been collected, repeat step 9 in Cell Extract Preparation
and Ribosome Footprinting with the remaining lysate to obtain the desired amount
of RNA.
b If < 1 μg of RPF RNA has been collected, repeat this purification procedure with
unused clarified lysate (from Ribosome Footprinting with TruSeq Ribo Profile
Nuclease on page 9) to obtain the desired amount of RNA.
8
Continue to Primary rRNA Depletion on page 13.
Sucrose Cushion
A sucrose cushion can be used as an alternative to the MicroSpin S-400 columns to
purify monosomes.1 Refer to Sucrose Cushion Purification of Monosomes on page 30.
TruSeq Ribo Profile (Mammalian) Kit
12
Purification of RPFs
a
Library Preparation
Primary rRNA Depletion
Consumables
Item
Quantity
Storage
Ribo-Zero Magnetic Gold Kit
(Human/Mouse/Rat)
1 kit
User
RNA Clean & Concentrator-5
Kit
1 kit
User
100% ethanol
Nuclease-Free Water
Supplied By
User
12/21 μl per
reaction
-20°C
Illumina
Procedure
1
Use 1–5 μg each of the RPF RNA and Total RNA samples that were prepared in
Purification of RPFs on page 11.
2
Follow the Ribo-Zero kit procedure with the following exceptions:
a Omit the 50°C incubation step (step 4 in Remove rRNA of the Ribo-Zero Kit
procedure).
NOTE
Omitting this step improves removal of small fragmented rRNA.
b
3
Do not purify the rRNA-depleted samples as stated in the Ribo-Zero procedure.
Instead, follow the purification procedure in the next step.
Purify both Ribo-Zero–treated samples (Total RNA and RPF RNA) using a modified
RNA Clean & Concentrator–5 Kit method:
a Bring samples up to 100 μl volume.
b Add RNA Binding Buffer and ethanol, and mix:
Reagent
RNA Binding
Buffer
100% Ethanol
c
d
Nuclease-Free
Water
13
RPF
Samples
200 μl
100 μl
450 μl
Continue with purification according to the manufacturer’s instructions.
Use the following elution amounts for the samples:
Reagent
e
Total RNA
Samples
100 μl
Total RNA
Samples
21 μl
RPF
Samples
11 μl
Expect to recover ~20 μl of the Total RNA sample. Keep on ice or store at -20°C
until ready for use in Fragmentation and End Repair on page 16.
Expect to recover ~10 μl of the RPF RNA sample.
Part # 15066016 Rev A
g
Quantify the samples by NanoDrop spectrophotometry. Expect to recover ~5%–
20% of input.
Proceed to the next section, PAGE Purification of RPFs.
PAGE Purification of RPFs
The RPFs are ~28-30 nt in length. The TruSeq Ribo Profile RNA Control that is provided
in the kit will be used as a size marker. It contains two oligos of 28 and 30 nt in length
(see Oligo Sequences on page 24).
CAUTION
Do not PAGE-purify the Ribo-Zero–treated Total RNA samples. Keep these samples on ice
or store at -20°C until ready for Fragmentation and End Repair on page 16.
Consumables
Item
Quantity
Storage
Supplied By
TruSeq Ribo Profile RNA
Control
5 μl per sample
-20°C
Illumina
Denaturing Gel Loading Dye
5/10 μl per sample
User
20/100 Oligo Ladder (1 ng/μl)
4 μl per lane
User
12% or 15%
polyacrylamide/7–8 M
urea/TBE gel
1 gel
User
SYBR Gold
User
5M Ammonium Acetate
40 μl per sample
-20°C
Illumina
10% SDS
2 μl per sample
-20°C
Illumina
Glycogen
2 μl per sample
-20°C
Illumina
100% isopropanol
700 μl per sample
User
80% ethanol, ice-cold, freshly
prepared
Nuclease-Free Water
User
-20°C
Illumina
Procedure
1
Prepare the TruSeq Ribo Profile RNA Control, samples, and ladder for PAGE.
a Add 5 μl of the TruSeq Ribo Profile RNA Control to a 0.5 ml microcentrifuge
tube. Add 5 μl of denaturing gel loading dye.
b Mix the RPF RNA samples (10 μl each) with 10 μl of denaturing gel loading dye
for a total volume of 20 μl).
c To prevent cross-contamination, prepare a ladder aliquot (4 μl of the 20/100
ladder, 1 μl of water, and 5 μl of denaturing gel loading dye) to load between
each RPF RNA sample or TruSeq Ribo Profile RNA Control.
2
Denature the samples and ladder by incubating at 95°C for 5 minutes. Place the
tubes immediately on ice. The TruSeq Ribo Profile RNA Control will be used as a
size marker.
TruSeq Ribo Profile (Mammalian) Kit
14
Library Preparation
f
3
Load 10 μl aliquots of each sample and ladder onto a 12% or 15% ureapolyacrylamide gel. Run the gel at 180 V until the Bromophenol Blue band reaches
bottom of the gel (~210 V for 1 hour; 180 V for 70 minutes). Store the remaining
samples at -20°C as backups.
4
Stain the gel with SYBR Gold and visualize the RNA using a dark-field
transilluminator (which emits a blue light).
NOTE
Chill SYBR Gold solution and stain the gel at 4°C to avoid diffusion of samples.
CAUTION
Do not use UV light to visualize gel bands. UV light damages nucleic acids, and library
preparation will fail.
5
For each sample, excise the gel slices corresponding to the 28 nt and 30 nt TruSeq
Ribo Profile RNA Control as a reference. Do this step even if footprints are not visible.
The ladder is overloaded to make size selection easier. However, this overloading
can oversaturate vision, making visualization of the RPFs challenging.
NOTE
Also excise the 28 nt and 30 nt RNA Control oligo bands. See Figure 4 in Optimizing
Nuclease Digestion to Generate RPFs on page 25 for an example.
6
Transfer each gel slice to a 0.5 ml microcentrifuge tube with a hole punched in the
bottom using a sterile 20 gauge needle, and close the tube cap. Place each 0.5 ml tube
inside a 1.5 ml tube and centrifuge for 2 minutes at ~12,000 × g in a microcentrifuge
to shred the gel slices.
NOTE
Samples may need to be centrifuged more than once to shred gel slices into the 1.5 ml
tube completely. This step shreds the gel and releases the RPFs from the gel
fragments.
7
Remove and discard the 0.5 ml tubes. To each 1.5 ml collection tube, add:
Reagent
Nuclease-Free Water
5M Ammonium Acetate
10% SDS
Volume (µl)
400
40
2
8
Gently rock the samples at room temperature for 1–2 hours or at 4°C overnight to
elute the RNA from the disrupted gel slices.
9
Use a wide-bore 1 ml pipette tip or trim the end of a 1 ml pipette tip to transfer the
slurry to 1.5 ml filter tubes (provided), and centrifuge for ~3 minutes at 2,000 × g to
separate disrupted gel pieces from the eluted RNA solution.
10 Use a large orifice 1 ml pipette tip or trim the end of a 1 ml pipette tip. Carefully
pipette the aqueous solution into new 1.5 ml tubes. To each tube, add 2 μl of
Glycogen and 700 μl of 100% isopropanol. Store at -20°C for > 1 hour.
11 Centrifuge the tubes at 4°C for 20 minutes at > 12,000 × g to pellet the RPF RNA.
Wash the RNA pellet with ice-cold 80% ethanol and air-dry.
12 Resuspend the pellet in the appropriate amount of Nuclease-Free Water:
15
Part # 15066016 Rev A
b
For the RPF RNA samples, resuspend the RNA pellet in 20 μl of Nuclease-Free
Water. Proceed to Fragmentation and End Repair on page 16 or freeze the samples
at -20°C.
For the TruSeq Ribo Profile RNA Control, resuspend the RNA pellet in 8 μl of
Nuclease-Free Water for use in 3′ Adapter Ligation on page 17. Store the samples
on ice or at -20°C until ready for use.
Fragmentation and End Repair
In this step, the Ribo-Zero–treated Total RNA sample is heat-fragmented. Both the
fragmented Total RNA and RPF RNA samples are then end-repaired to prepare them for
the 3′ adapter ligation step (3′ Adapter Ligation on page 17). RNA end-repair is performed
using TruSeq Ribo Profile PNK in the absence of ATP.
Consumables
Item
Quantity
Storage
Supplied By
TruSeq Ribo Profile PNK
Buffer
7.5 μl per reaction
-20°C
Illumina
TruSeq Ribo Profile PNK
3 μl per reaction
-20°C
Illumina
RNA Clean & Concentrator–
5 Kit
1 kit
User
100% ethanol
User
80% ethanol, ice-cold, freshly
prepared
User
Nuclease-Free Water
-20°C
Illumina
Procedure
1
To a 20 μl aliquot each of the Total RNA and RPF RNA samples on ice, add 7.5 μl of
TruSeq Ribo Profile PNK Buffer.
SAFE STOPPING POINT
Hold the RPF RNA samples on ice for use in step 3 of this procedure.
2
Heat only the Total RNA sample at 94°C for 25 minutes. Place on ice or hold at 4°C.
3
To the heat-fragmented Total RNA and RPF RNA sample, add:
Reagent
Nuclease-Free Water
TruSeq Ribo Profile PNK
Total Volume per Sample
Volume (µl)
44.5
3.0
75.0
4
Mix well. Incubate the samples at 37°C for 1 hour.
5
Adjust each sample volume to 100 μl with 25 μl of Nuclease-Free Water. Purify the
samples using a modified RNA Clean & Concentrator–5 Kit method:
a Add 200 μl of RNA Binding Buffer and 450 μl of 100% ethanol. Mix well.
b Continue with the purification according to the manufacturer’s instructions
c Elute the samples with 11 μl of Nuclease-Free Water. Expect to recover ~8 μl.
TruSeq Ribo Profile (Mammalian) Kit
16
Library Preparation
a
SAFE STOPPING POINT
Optional: Store the samples at -20°C or lower, for up to 7 days.
6
Proceed to the next section, 3′ Adapter Ligation.
3′Adapter Ligation
It is useful to include both positive and negative controls. The positive control will also
be used in downstream PAGE analysis.
} CtrlP: Positive Control = PAGE-purified TruSeq Ribo Profile RNA Control from step
12 in PAGE Purification of RPFs on page 14.
} CtrlN: Negative Control = no RNA (mock reaction; use 8 μl of Nuclease-Free Water).
Consumables
Item
Quantity
Storage
Supplied By
TruSeq Ribo Profile 3′ Adapter
1 μl per reaction
-20°C
Illumina
TruSeq Ribo Profile RNA
Control
1 μl per reaction
-20°C
Illumina
TruSeq Ribo Profile Ligation
Buffer
3.5 μl per reaction
-20°C
Illumina
TruSeq Ribo Profile Ligase
1.5 μl per reaction
-20°C
Illumina
TruSeq Ribo Profile AR
Enzyme
2 μl per reaction
-20°C
Illumina
DTT (100 mM)
1 μl per reaction
-20°C
Illumina
Procedure
1
Keep the RNA samples from Fragmentation and End Repair on page 16 and the
controls on ice. Add 1.0 μl of the TruSeq Ribo Profile 3′ Adapter. In a thermal cycler
with heated lid, heat-denature the samples for 2 minutes at 65°C, then hold at 4°C.
2
While the denaturing reaction is proceeding, prepare the ligation mastermix:
Reagent
TruSeq Ribo Profile Ligation Buffer
100 mM DTT
TruSeq Ribo Profile Ligase
Total Volume per Sample
3
Volume (µl)
3.5
1.0
1.5
6.0
Add 6 μl of the ligation mastermix to each of the denatured RNA samples. Mix
thoroughly by pipetting several times. Centrifuge briefly to consolidate the sample at
the bottom of the tube. Incubate at room temperature (23°C) for 2 hours.
SAFE STOPPING POINT
Optional: Store the samples at -20°C or lower.
4
17
Add 2 μl of TruSeq Ribo Profile AR Enzyme to each reaction and mix thoroughly by
pipetting. Incubate at 30°C for 2 hours.
Part # 15066016 Rev A
Consumables
Item
Quantity
Storage
Supplied By
TruSeq Ribo Profile RT
Reaction Mix
4.5 μl per reaction
-20°C
Illumina
EpiScript RT
1 μl per reaction
-20°C
Illumina
TruSeq Ribo Profile
Exonuclease
1 μl per reaction
-20°C
Illumina
TruSeq Ribo Profile RNase
Mix
1 μl per reaction
-20°C
Illumina
DTT (100 mM)
1.5 μl per reaction
-20°C
Illumina
Nuclease-Free Water
6 μl per reaction
-20°C
Illumina
Procedure
1
For each reaction, prepare the reverse transcription mastermix on ice:
Reagent
TruSeq Ribo Profile RT Reaction Mix
100 mM DTT
Nuclease-Free Water
EpiScript RT
Total Volume per Sample
Volume (µl)
4.5
1.5
6.0
1.0
13.0
2
Add 13 μl of the reverse transcription mastermix to each reaction and mix well by
pipetting.
3
Incubate the reactions for 30 minutes at 50°C in a thermal cycler with a heated lid.
4
Add 1 μl of TruSeq Ribo Profile Exonuclease to each reaction and incubate:
• 37°C for 30 minutes
• 80°C for 15 minutes
• Hold at 4°C
SAFE STOPPING POINT
Optional: Store the samples at -20°C or lower.
5
Add 1 μl of TruSeq Ribo Profile RNase Mix to each reaction. Mix and incubate for 5
minutes at 55°C. Place the reactions on ice or hold at 4°C.
TruSeq Ribo Profile (Mammalian) Kit
18
Library Preparation
Reverse Transcription
PAGE Purification of cDNA
Consumables
Item
Quantity
RNA Clean & Concentrator–5
Kit
1 kit
Storage
Supplied By
User
100% ethanol
User
80% ethanol, ice-cold,
freshly prepared
User
Nuclease-Free Water
40 μl per sample
-20°C
20/100 oligo ladder
4 μl per lane
User
10% polyacrylamide/7–8M
urea/TBE gel
1 gel
User
Denaturing gel loading dye
5/10 μl per sample
User
Dark field transilluminator
1
User
SYBR Gold
Illumina
User
5M ammonium acetate
40 μl per sample
-20°C
Illumina
10% SDS
2 μl per sample
-20°C
Illumina
Glycogen
2 μl per sample
-20°C
Illumina
100% isopropanol
700 μl per sample
User
Procedure
1
Add 18 μl of Nuclease-Free Water to each reaction, bringing the final volume to
50 μl.
2
Purify the samples using a modified RNA Clean & Concentrator–5 Kit method:
a Add 100 μl of RNA Binding Buffer and 150 μl of 100% ethanol. Mix well.
b Continue with purification according to the manufacturer’s instructions
c Elute the samples with 11 μl of Nuclease-Free Water. Expect to recover ~10 μl.
3
Gel-purify the cDNA (see PAGE Purification of cDNA on page 28).
cDNA Circularization
Consumables
19
Item
Quantity
Storage
Supplied By
TruSeq Ribo Profile CL
Reaction Mix
4 μl per reaction
-20°C
Illumina
Part # 15066016 Rev A
Quantity
Storage
Supplied By
ATP
2 μl per reaction
-20°C
Illumina
MnCl2
2 μl per reaction
-20°C
Illumina
CircLigase
2 μl per reaction
-20°C
Illumina
Procedure
1
Prepare the CircLigase mastermix on ice:
Reagent
TruSeq Ribo Profile CL Reaction Mix
ATP
MnCl2
CircLigase
Total Volume per Sample
2
Volume (µl)
4.0
2.0
2.0
2.0
10.0
Add 10 μl of the CircLigase mastermix to each reaction. Mix the tubes gently and
centrifuge briefly. Incubate the reactions at 60°C for 2 hours, then place on ice or hold
at 4°C.
PCR Amplification
Too much template or too many PCR cycles can result in overamplification—the
appearance of higher than expected molecular weight bands, smeared PCR products,
and adapter-dimer–derived products. Examples of overamplified samples can be seen in
Overamplifying RPF cDNA on page 27.
For most samples, 1-5 μl of the circularized cDNA from cDNA Circularization on page 19
and 9 PCR cycles will typically yield sufficient amounts of the correct PCR product.
A good starting strategy is to set up two PCRs for each sample. For one PCR , use 5 μl of
1:5 dilution (with water) of the circularized cDNA as template. For the second PCR, use 5
μl of undiluted circularized cDNA.
Before beginning this procedure:
} Choose one of the TruSeq Ribo Profile Index PCR Primers (provided) for use as the
Reverse PCR Primer.
} If pooling libraries for sequencing later, check the indexes for color balance during
sequencing. See TruSeq Ribo Profile Index PCR Primers (1-12) on page 24 for
recommendations on index combinations for sequencing.
NOTE
The PCR is optimized for use with 2X Phusion MasterMix.
Consumables
Item
Quantity
Storage
Supplied By
TruSeq Ribo Profile Forward
PCR Primer
2 μl per reaction
-20°C
Illumina
TruSeq Ribo Profile (Mammalian) Kit
20
Library Preparation
Item
Item
Quantity
Storage
Supplied By
TruSeq Ribo Profile Index
PCR Primers
2 μl per reaction
-20°C
Illumina
2X Phusion MasterMix
25 μl per reaction
-20°C
User
AMPure XP System
1 kit
User
100% ethanol
User
80% ethanol, ice-cold, freshly
prepared
User
Nuclease-Free Water
-20°C
Illumina
Native gel loading dye
containing Bromophenol Blue
1 μl per sample
User
20 bp Ladder
6 μl per lane
User
8% native
polyacrylamide/TBE gel
1 gel
User
SYBR Gold
User
Procedure
1
Prepare the PCR mastermix. For each sample, combine on ice:
Reagent
Nuclease-Free Water
TruSeq Ribo Profile Forward PCR Primer
TruSeq Ribo Profile Index PCR Primer of choice
2X Phusion Master Mix
Total Volume per Sample
21
Volume (µl)
16
2
2
25
45
2
Add 45 μl of the PCR Master Mix to each of the 5 μl samples of 1:5 diluted and
undiluted cDNA. Mix the tubes well.
3
Place all tubes in a thermal cycler and run the following program:
• 98°C for 30 seconds
• 9 cycles of
— 94°C for 15 seconds
— 55°C for 5 seconds
— 65°C for 10 seconds
• Hold at 4°C
4
Purify the PCR products using 90 μl (1.8X) of Agencourt AMPure XP beads
according to the manufacturer’s instructions. Elute the libraries with 16 μl of
Nuclease-Free Water or 10 mM Tris-HCl (pH 8.0).
5
Remove 2.5 μl aliquots from each sample to a new tube at room temperature. Add
1.5 μl of water and 1 μl of 6X native gel loading dye containing Bromophenol Blue.
6
Load 5 μl of the PCR product aliquots from step 5 of this procedure and a 20 bp
ladder onto a 8% native polyacrylamide gel in 1X TBE. Run the gel at 200 V until the
Bromophenol Blue reaches the bottom of the gel (~83 V for 1 hour; 200 V for 25
Part # 15066016 Rev A
7
The size distribution of the library can be characterized alternatively using the
Agilent High Sensitivity DNA Assay.
8
The expected size of the PCR-amplified library is 140–160 bp. Select the tubes that
contain the correct PCR product for purification and sequencing.
NOTE
If adapter product (~113 bp) is observed (see Figure 3), PAGE-purify the desired product
using an 8% native polyacrylamide gel (see PAGE Purification of PCR Products on page 27).
If the desired product (~140–160 bp) is only visible weakly, but too weakly to purify, you
can do the following, one at a time - until you have sufficient cDNA for your protocol.
a. Amplify leftover ciruclarized material from PCR amplification and pool multiple
PCR reactions.
b. PAGE-purify the backup linear cDNA from step in PAGE Purification of cDNA . See
PAGE Purification of cDNA on page 28.
c. For RPF samples, PAGE purify RPF RNA sample back-ups from PAGE Purification of
RPFs on page 14.
TruSeq Ribo Profile (Mammalian) Kit
22
Library Preparation
minutes). Stain the gel with SYBR Gold and detect under a dark field
transilluminator.
9
Proceed to sequencing. See Sequencing a TruSeq Ribo Profile Library on page 25.
Figure 3 BioAnalyzer Profiles of TruSeq Libraries and Optional Gel Purification
A
B
TruSeq libraries were prepared from 2 ribosome-protected mRNA samples, amplified for 9
cycles of PCR, and purified using AMPure Beads. Samples were analyzed by the Agilent
High Sensitivity DNA Assay.
A
B
A "good" sample. The ~140–160 bp peak is the expected size range and no further
purification is needed.
A sample containing an excessive amount of adapter-dimer amplified product
(~120 bp) relative to the desired product (~140–160 bp). This sample should be purified
further (see PAGE Purification of PCR Products on page 27).
Reference
1
23
Ingolia, NT. Genome-wide translational profiling by ribosome footprinting. Methods
Enzymol 2010;470:119-142.
Part # 15066016 Rev A
Appendices
Appendices
Oligo Sequences
TruSeq Ribo Profile RNA Control Oligos (2)
5′ NNGUACACGGAGUCGACCCGCAACGCNN 3′ (28 nt)
5′ NNGUACACGGAGUCAAGACCCGCAACGCNN 3′ (30 nt)
TruSeq Ribo Profile 3′ Adapter
5′ AGATCGGAAGAGCACACGTCT 3′
TruSeq Ribo Profile Forward PCR Primer
5′ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACG 3′
TruSeq Ribo Profile Index PCR Primers (1-12)
A TruSeq Ribo Profile Index PCR Primer functions as the Reverse PCR Primer during
PCR amplification. The sequence of each Index PCR Primer is:
5’ CAAGCAGAAGACGGCATACGAGAT NNNNNN GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 3’
where NNNNNN is the reverse complement of the index sequence generated during
sequencing (see Table 6).
Table 6 Index Sequence Generated During Sequencing
Primer
Index Sequence Generated
Index 1 PCR Primer
Index 2 PCR Primer
Index 3 PCR Primer
Index 4 PCR Primer
Index 5 PCR Primer
Index 6 PCR Primer
Index 7 PCR Primer
Index 8 PCR Primer
Index 9 PCR Primer
Index 10 PCR Primer
Index 11 PCR Primer
Index 12 PCR Primer
5′-ATCACG-3′
5′-CGATGT-3′
5′-TTAGGC-3′
5′-TGACCA-3′
5′-ACAGTG-3′
5′-GCCAAT-3′
5′-CAGATC-3′
5′-ACTTGA-3′
5′-GATCAG-3′
5′-TAGCTT-3′
5′-GGCTAC-3′
5′-CTTGTA-3′
i7 Index ID for Entry Into
the IEM Sample Sheet Wizard
A001
A002
A003
A004
A005
A006
A007
A008
A009
A010
A011
A012
Table 7 [Recommended Index Combinations for Sequencing Library Pools]
Duplex (two indices)
Index 06 and Index 12
(Perfect base color balance)
Triplex (three indices)
Index 04 and Index 08
Add one: Index 01, 02, 05, 06, 09, or 10
Triplex (three indices)
Index 07 and Index 11
Add one: Index 01, 02, 05, 06, 09, or 10
Triplex (three indices)
Index 02 and Index 09
Add one: Index 03, 04, 07, 08, 11, or 12
Four or more indices
Index 06 and Index 12
Add any other indexes
Pooling Multiplexed TruSeq Ribo Profile Libraries
Illumina sequencing systems use a green laser to read G/T nucleotides and a red laser to
read A/C nucleotides. At least 1 nucleotide for each color channel must be read in each
sequencing cycle to ensure proper registration. It is important to maintain color balance
for each base of the index read being sequenced, otherwise index read sequencing could
TruSeq Ribo Profile (Mammalian) Kit
24
fail due to registration failure. Table 8 demonstrates proper and improper pooling
strategies.
Table 8 Pooling Strategies for Indexes
Good
Bad
04
TGACCA
09
GATCAG
01
ATCACG
07
CAGATC
05
ACAGTG
10
TAGCTT
02
CGATGT
08
ACTTGA
06
GCCAAT
11
GGCTAC
03
TTAGGC
09
GATCAG
07
CAGATC
12
CTTGTA
04
TGACCA
10
TAGCTT
√√√√√√
√√√√√√
√xx√√√
√xx√√√√
√ = signal in both color channels; x = signal missing in one color channel
} Pooling and sequencing TruSeq Ribo Profile libraries containing indexes 4, 5, 6, 7 or
indexes 9, 10, 11, 12 results in successful registration due to the presence of both red
and green signals when sequencing each of the 6 nucleotides of the indexes.
} Pooling and sequencing TruSeq Ribo Profile libraries containing indexes 1, 2, 3, 4 or
indexes 7, 8, 9, 10 can result in a registration failure due to lack of both green and
red color signals when sequencing nucleotides 2 and 3 of the indexes.
Sequencing a TruSeq Ribo Profile Library
Purified, adapter-tagged TruSeq Ribo Profile libraries are compatible with Illumina
sequencing systems. The sequence generated by Read 1 is that of the sense strand of the
original RNA. Recommended sequencing read length for TruSeq Ribo Profile libraries is
51 cycles (Read 1 only or Read 1 plus Index Read); Bioanalyzer analysis of libraries
shows approximately 150 bp fragments (see BioAnalyzer Profiles of TruSeq Libraries and
Optional Gel Purification on page 23 ) with the actual insert size at approximately 28–30
nt.
For Index Primer sequences and pooling suggestions, see TruSeq Ribo Profile Index PCR
Primers (1-12) on page 24.
NOTE
To use Illumina Experiment Manager (IEM), select TruSeq LT to access the appropriate
barcodes.
TruSeq Ribo Profile libraries have sufficient complexity for single sample per lane–
sequencing. Multiplexing is optional and therefore generous PhiX DNA spike-ins (to
prevent registration issues) are not needed. PhiX DNA may be added to monitor error
rates during sequencing, if desired.
The minimum recommended number of reads for each sample library is 40 million. For
guidance on analyzing sequencing data, please refer to the TruSeq Ribo Profile
Bioinformatics User Guide.
Optimizing Nuclease Digestion to Generate RPFs
RPFs can be contaminated with partially degraded tRNA fragments. Therefore, it may be
desirable to optimize the TruSeq Ribo Profile Nuclease digestion step in order to
maximize RPF recovery and minimize contamination. TruSeq Ribo Profile Nuclease is
provided at a concentration of 10 U/μl.
1
25
Set up a titration series of TruSeq Ribo Profile Nuclease using individual aliquots of
cell lysate. For example, to 4 separate 200 μl aliquots of the cell lysate, add 0.3 μl
Part # 15066016 Rev A
2
Incubate the reactions at room temperature for 45–60 minutes.
3
Stop the reactions by adding 15 μl of SUPERase•In RNase Inhibitor and chill the
samples on ice.
4
Purify 100 μl of each reaction using the MicroSpin S-400 columns and extract RNA
as described in step 1 through step 7 of the standard procedure (MicroSpin S-400
Columns). Freeze the remaining sample in liquid nitrogen and store at -80°C.
5
Prepare the RNA Control, RPF samples, and a ladder for PAGE.
a Add 1 μl of the TruSeq Ribo Profile RNA Control from the kit to a 0.5 ml tube.
Add 9 μl of denaturing gel loading dye.
b For each of the resuspended RPF samples from step 4 of this procedure, mix 250
ng of the sample with denaturing gel loading dye up to 10 μl final volume.
c Prepare a ladder aliquot (2 μl of 20/100 ladder, 3 μl of water, and 5 μl of
denaturing gel loading dye).
6
Heat all samples to 95°C for 5 minutes. Place the tubes immediately on ice. The
TruSeq Ribo Profile RNA Control (28 nt and 30 nt) oligo will be used for a size
marker.
7
Load 10 μl aliquots of each sample onto a 12% or 15% urea-polyacrylamide gel. Run
the gel at 180 V until the Bromophenol Blue band reaches the bottom of the gel
(~210V for 1 hour; 180 V for 70 minutes). Stain the gel with SYBR Gold. Visualize
the gel with UV light or a dark-field transilluminator.
8
Continue with Ribo-Zero rRNA removal (Primary rRNA Depletion on page 13) with
the titrated sample from step 4 of this procedure that has a distinct RPF band,
similar to the region of the 60 U sample shown in Figure 4.
Figure 4 Denaturing PAGE Analysis of Nuclease Titration
The effect of increasing amounts of TruSeq Ribo Profile Nuclease on RPF recovery: 60 units
of nuclease was optimal to observe RNase-resistant material corresponding to the size of
RPFs (dotted section).
TruSeq Ribo Profile (Mammalian) Kit
26
Appendices
(3 U), 1 μl (10 U), 3 μl (30 U), and 6 μl (60 U) of TruSeq Ribo Profile Nuclease,
respectively.
Example: 50 A260/ml lysate × 0.2 ml lysate × 5 U/A260 nuclease = 50 U nuclease
Overamplifying RPF cDNA
Overamplification of the RPFs in PCR Amplification on page 20 can lead to the
appearance of higher than expected amplicons, smeared amplicons, and adapter-dimer
products. In the example shown (Figure 5), 9 cycles of PCR yielded the optimal amount
of the expected PCR product.
Figure 5 Effect of PCR Cycle Number on Product Size
"+" libraries were prepared from the 28 nt Control RNA. In "-" negative control (no RNA)
libraries, the band appearing at ~120 bp in 12 and 15 cycles is adapter-dimer. S1 and S2 are
RPF samples.
PAGE Purification of PCR Products
1
Prepare samples, positive control, and ladders for PAGE.
a For each ~25 μl PCR sample to be purified, and the amplified positive control,
add 6 μl of native gel loading dye containing Bromophenol Blue.
b Separate samples with a 5 μl aliquot of the 20 bp ladder, or a 1 μl aliquot of
diluted native gel loading dye containing Bromophenol Blue and 5 μl water.
2
Load ~15 μl per lane (split samples split across 2 lanes if necessary) of a 8% native
polyacrylamide gel in 1X TBE, separating samples as described in step 1b of this
procedure.
3
Run the gel at 200 V until the Bromophenol Blue dye reaches the bottom (~83 V for
1 hour; 200 V for 25 minutes). Stain the gel with SYBR Gold and visualize using a
dark field transilluminator.
NOTE
Chill SYBR Gold solution and stain the gel at 4°C to avoid diffusion of samples.
CAUTION
Do not use UV light to visualize gel bands. UV light damages DNA, and library
preparation will fail.
4
Excise the uppermost band (~140-160 bp), being careful to avoid the adapter-dimer
(~113 bp).
5
Transfer each gel slice to a 0.5 ml microcentrifuge tube with a hole punched in the
bottom using a sterile 20 gauge needle, and close the tube cap. Place each 0.5 ml tube
inside a 1.5 ml tube and centrifuge for 2 minutes at ~12,000 × g in a microcentrifuge
to shred the gel slices.
NOTE
Samples may need to be centrifuged more than once to shred gel slices into the 1.5 ml
tube completely. This step shreds the gel and releases the PCR products from the gel
fragments.
6
27
Remove and discard the 0.5 ml tubes. To each 1.5 ml collection tube, add:
Part # 15066016 Rev A
Volume (µl)
400
40
2
7
Gently rock the samples at 37°C for 1–2 hours or at room temperature overnight to
elute the PCR products from the disrupted gel slices.
8
Transfer the slurry to new 1.5 ml filter tubes and centrifuge for ~3 minutes at
2,000 × g to separate the disrupted gel pieces from the eluted PCR product solution.
9
Use a wide-bore 1 ml pipette tip or trim the end of a 1 ml pipette tip. Carefully
pipette the aqueous solution into new 1.5 ml tubes. To each tube, add 2 μl of
Glycogen and 700 μl of 100% isopropanol. Store at -20°C for > 1 hour.
CAUTION
Use the Glycogen that is provided in the kit. Do not use glycogen with added dye,
such as GlycoBlue, that can interfere with quantification of the nucleic acid.
10 Centrifuge the tubes at 4°C for 20 minutes at > 12,000 × g to pellet the PCR product.
Wash the pellet with ice-cold 80% ethanol and air-dry.
11 Resuspend the pellet in 15 μl of Nuclease-Free Water or 10 mM Tris-HCl (pH 8.0).
Pool tubes of the same sample together (sample volume = 30 μl).
12 Check 1 μl of each sample on the Bioanalyzer using the Agilent High Sensitivity
DNA Assay.
13 Proceed to sequencing. See Sequencing a TruSeq Ribo Profile Library on page 25 for
sequencing information.
PAGE Purification of cDNA
Purification of cDNA can improve amplification of the desired products in PCR
Amplification on page 20
1
Prepare the cDNA samples (10 μl) from step in PAGE Purification of cDNA and a
ladder for PAGE.
a Add 10 μl of denaturing gel loading dye to each cDNA sample and the Control
RNA.
b Separate each sample or TruSeq Ribo Profile RNA Control with an aliquot of the
20/100 Oligo Ladder (4 μl ladder, 1 μl water, and 5 μl denaturing loading dye)
to avoid cross-contamination.
2
Heat the samples to 95°C for 5 minutes and place on ice.
3
Load 10 μl of each sample onto a single lane of a 10% polyacrylamide/7–8 M
urea/TBE gel. Load a second lane with the remaining 10 μl of the sample, or store it
at -20°C as backup.
4
Run the gel at 180 V until the Bromophenol Blue dye completely migrates out of the
gel (~180 V for 1 hour). Stain the gel with SYBR Gold and visualize using a darkfield transilluminator.
NOTE
Chill SYBR Gold solution and stain the gel at 4°C to avoid diffusion of samples.
TruSeq Ribo Profile (Mammalian) Kit
28
Appendices
Reagent
Nuclease-Free Water
5M Ammonium Acetate
10% SDS
CAUTION
Do not use UV light to visualize gel bands. UV light damages nucleic acids, and library
preparation will fail.
5
Excise the gel slices. Do this step even if bands are not visible. The ladder is
overloaded to make size selection easier. However, this overloading can oversaturate
vision, making visualization of the cDNA challenging.
a For the Ribo-Zero–treated Total RNA samples, excise bands corresponding to
~80-100 nt.
b For the RPF samples, and CtrlN and CtrlP controls, excise bands corresponding
to ~70-80 nt.
NOTE
In the CtrlP sample a distinct cDNA product migrating at 73–75 nt should be
visible and excised for use as a positive control. It is also a helpful size marker for
comparison to the RPF samples. Also excise the same region for the CtrlN sample
for use as the negative control. The size ranges are higher than the previous gel
purification due to the addition of the 3′ Adapter and RT Primer. See Figure 6 for
examples.
CtrlP and sample cDNA at 70-100 nt may or may not be visible on the
purification gel. Continue to excise gel regions within this range. The ladder is
overloaded to make size selection easier but tends to make visualization of the
cDNA bands difficult.
6
Transfer each gel slice to a 0.5 ml microcentrifuge tube with a hole punched in the
bottom using a sterile 20 gauge needle, and close the tube cap. Place each 0.5 ml tube
inside a 1.5 ml tube and centrifuge for 2 minutes at ~12,000 × g in a microcentrifuge
to shred the gel slices.
NOTE
Samples may need to be centrifuged more than once to shred gel slices into the 1.5 ml
tube completely. This step shreds the gel and releases the cDNA from the gel
fragments.
7
Remove and discard the 0.5 ml tubes. To each 1.5 ml collection tube, add:
Reagent
Nuclease-Free Water
5M Ammonium Acetate
10% SDS
Volume (µl)
400
40
2
8
Gently rock the samples at 37°C for 1–2 hours or at room temperature overnight to
elute the cDNA from the disrupted gel slices.
9
Transfer the slurry to new 1.5 ml filter tubes and centrifuge for ~3 minutes at
2,000 × g to separate the disrupted gel pieces from the eluted cDNA solution.
10 Use a wide-bore 1 ml pipette tip or trim the end of a 1 ml pipette tip. Carefully
pipette the aqueous solution into new 1.5 ml tubes. To each tube, add 2 μl of
Glycogen and 700 μl of 100% isopropanol. Store at -20°C for > 1 hour.
11 Centrifuge the tubes at 4°C for 20 minutes at > 12,000 × g to pellet the cDNA. Wash
the pellet with ice-cold 80% ethanol and air-dry.
12 Resuspend the pellet in 10 μl of Nuclease-Free Water.
13 Proceed to cDNA Circularization on page 19 or store the samples at -20°C.
29
Part # 15066016 Rev A
Appendices
Figure 6
PAGE Purification of cDNA
The cDNA reactions from the CtrlN (lanes 2 and 3) and CtrlP (lanes 5 and 6) samples were
separated by PAGE. To purify, excise gel regions corresponding to sizes ~70–80 nt for RPF
samples and the CtrlN and CtrlP samples (see dotted lines) and extract the cDNA as
described in the protocol. The 73 and 75 nt cDNA products will likely be observed in the
CtrlP lanes corresponding to the 28 nt and 30 nt Control RNA. For Ribo-Zero–treated Total
RNA samples, excise the size range ~80–100 nt and extract the cDNA as described in the
protocol.
Sucrose Cushion Purification of Monosomes
1
For each sample, prepare 1 ml of Sucrose Cushion Buffer:
Reagent
5X Mammalian Polysome Buffer
Sucrose
SUPERase•In (20 U/μl)
MgCl2 (25 mM)
DTT (100 mM)
Cycloheximide (50 mg/ml)
Amount
200 μl
0.5 g
5 μl
140 μl
5 μl
2 μl
Adjust the final volume to 1 ml with Nuclease-Free Water.
2
Carefully layer 200 μl of the TruSeq Ribo Profile Nuclease/SUPERase•In–treated
lysate over 1 ml of ice-cold Sucrose Cushion Buffer in an appropriate ultracentrifugecompatible tube.
3
Pellet the ribosomes by centrifugation at 4°C for 4 hours at 70,000 rpm in a TLA-110
rotor.
4
Remove the supernatant and resuspend the RNA pellets in 100 μl of Nuclease-Free
Water or 10 mM Tris-HCl (pH 7.0). Add 10 μl of 10% SDS. This material will be
used as RPF RNA. Store the samples at -80°C, or proceed to the next step.
5
Purify both Total RNA and RPF RNA samples using a modified RNA Clean and
Concentrator–25 Kit method:
TruSeq Ribo Profile (Mammalian) Kit
30
a
Add RNA Binding Buffer and ethanol and mix:
Reagent
RNA Binding
Buffer
100% Ethanol
b
c
31
Total RNA
Samples
220 μl
RPF
Samples
220 μl
220 μl
495 μl
Continue with purification according to the manufacturer’s instructions.
Elute the samples with 26 μl of Nuclease-Free Water. Expect to recover ~25 μl.
6
Quantify by NanoDrop spectrophotometry. You will need about 1–5 μg of Total RNA
and 1–5 μg of RPF RNA for the Ribo-Zero rRNA removal treatment in Primary rRNA
Depletion on page 13.
a If < 1–5 μg of Total RNA has been collected, repeat step 9 in Cell Extract
Preparation and Ribosome Footprinting with the remaining lysate to obtain the
desired amount of RNA.
b If < 1–5 μg of RPF RNA has been collected, repeat Ribosome Footprinting with
TruSeq Ribo Profile Nuclease on page 9 and MicroSpin S-400 Columns on page 11 or
Sucrose Cushion Purification of Monosomes on page 30 with unused clarified lysate
to obtain the desired amount of RNA.
7
Continue to Primary rRNA Depletion on page 13.
Part # 15066016 Rev A
For technical assistance, contact Illumina Technical Support.
Table 9 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 10 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
Other countries
Contact Number
800.874909
0800.0223859
0800.451.650
800.16836
900.812168
020790181
0800.563118
0800.917.0041
+44.1799.534000
Safety Data Sheets
Safety data sheets (SDSs) are available on the Illumina website at
support.illumina.com/sds.html.
Product Documentation
Product documentation in PDF is available for download from the Illumina website. Go
to support.illumina.com, select a product, then select Documentation & Literature.
TruSeq Ribo Profile (Mammalian) Kit
Technical Assistance
Technical Assistance
*15066016*
Part # 15066016 Rev A
Illumina
San Diego, California 92122 U.S.A.
+1.800.809.ILMN (4566)
+1.858.202.4566 (outside North America)
[email protected]
www.illumina.com