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Date:______________________
PI/Project/Sample Name(s):__________________________________________
Illumina TruSeq RNA Sample Library Preparation
Low Throughput (LT) Protocol, Part # 15015050 Rev. A
Modified by USC Epigenome Center v 110330
Preparations:
Use only RNAse free diH20
Prior to starting decontaminate with RNAse Zap Spray everything to be used!
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RNA Sample QC:
Thaw samples on ice, vortex & centrifuge, keep on ice
Assess sample quality via Bio-Rad Experion or Agilent Bioanalyzer RNA Chip
Assess sample quantity via Nanodrop (ng/µl) – see Nanodrop protocol
Calculate stock volume needed to obtain 2µg (0.1-4µg allowable) to start
V(stock) = 2000ng / C(nanodrop) ng/µl
Calculate volume RNAse free diH20 needed to bring total up to 50µl.
Sample
Nanodrop
(ng/µl)
Volume of Sample
Stock for 2µg
RNAse free diH20 volume
to total 50µl
Preparations:
§ Pre-program Thermocycler with the following programs:
a. mRNA Denaturation: 65oC for 5 m, 10oC ∞
b. mRNA Elution: 80oC for 2 m, 25oC ∞
c. Elution-Fragment-Prime: 94oC for 8 m, 10oC ∞
d. 1st Strand: 25oC for 10 m→42oC for 50 m→70oC for 15 m→10oC ∞
e. 2nd Strand: 16oC for 60 m→25oC ∞
~6 hr to 1st safe stop in protocol
DO NOT PROCEED WITHOUT SUFFICIENT TIME!
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Preheat thermocycler lid to 100oC
if refrigerated, set centrifuge to -15oC to -25oC
Thaw samples & reagents, vortex, centrifuge & keep on ice!
- Bead Binding Buffer
- Bead Washing Buffer
- Elution Buffer
Briefly vortex & centrifuge each once thawed
Bring RNA Purification Beads to RT (from 4oC), mix thoroughly
mRNA-Seq Page 1 of 7.
Date:______________________
PI/Project/Sample Name(s):__________________________________________
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mRNA to cDNA conversion – Purify/Fragment mRNA:
Vortex RNA Purification Beads to mix thoroughly
Add 50µl RNA Purificatoin beads to 50µl sample, pipet 6x gently to mix, cap & flick mix
Thermocycle a. mRNA Denature (65oC for 5 m, 10oC ∞)
When reaches 10oC remove from thermocycler, incubate @ RT 5 m
Condense beads on magnet ~3 m
Without disturbing bead pellet - Remove & discard supernatant
Add 200µl Bead Washing Buffer & Condense beads on magnet ~3 m
Remove & discard supernatant - DO NOT DISTURB BEAD PELLET!
Add 50µl Elution Buffer, pipet 6x gently to mix, cap & flick mix
Thermocycle b. mRNA Elution (80oC for 2 m, 25oC ∞)– keep at RT
Add 50µl Bead Binding Buffer, pipet 6x gently to mix, cap & flick mix
incubate @ RT 5 m, then condense bead pellet on magnet ~3 m
§ Thaw Elute-Prime-Fragment Mix
§ leave sample on magnet to minimize disturbing condensed bead pellet
Remove & discard supernatant - DO NOT DISTURB BEAD PELLET!
Add 200µl Bead Washing Buffer & Condense beads on magnet 5 m
Remove & discard supernatant - DO NOT DISTURB BEAD PELLET!
Remove from magnet
Add 19.5µl Elute-Prime-Fragment Mix, pipet 6x to gently elute sample, cap & flick mix
Thermocycle c. Elution-Frag-Prime (94oC for 8 m, 10oC ∞)
§ Re-freeze Elute-Prime-Fragment Mix immediately after use
Remove from thermocycle as soon as reaches 10oC, centrifuge briefly
Proceed immediately to Synthesize 1st Strand cDNA – place on magnet
§ Leave sample on magnet to minimize disturbing condensed bead pellet
Synthesize 1st Strand cDNA
Preparations:
§ Thaw 1 tube of 1st Strand Master Mix & Superscript III, vortex briefly, centrifuge
§ Add 50µl Superscript III to 1 tube 1st Strand Master Mix (1:7 ratio if mixing less)
§ Mix gently, centrifuge briefly, label as “SS added on date”, keep on ice
§ If more than 6 freeze/thaw cycles anticipated aliquot into smaller volumes
o Leave sample on magnet, DO NOT DISTURB condensed bead pellet
o Transfer supernatant (fragmented, primed mRNA) to new labeled tube
o Add 8µl 1st Strand MM + SuperScript III Mix, pipet 6x gently to mix, cap & flick mix
§ Re-freeze 1st Strand MM + SS III Mix immediately after use
o Thermocycle d. 1st Strand (25oC 10 m→42oC 50 m→70oC 15 m→10o
mRNA-Seq Page 2 of 7.
Date:______________________
PI/Project/Sample Name(s):__________________________________________
cDNA – Synthesize 2nd Strand:
Preparations:
§ Thaw 1 tube 2nd Strand Master Mix & centrifuge briefly
§ Thaw Re-Suspension Buffer
§ Vortex & centrifuge once thawed
§ invert AMPure XP Bead Stock (from 4oC) to mix thoroughly, pipet sufficient
working aliquot for protocol, return stock to 4oC
§ vortex working aliquot of beads vigorously to thoroughly mix
§ Review & follow Protocol for Handling AMPure XP Beads
§ Chill thermocycler to 16oC (e. 2nd Strand recipe)
§ Mix fresh 70% EtOH
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Add 25µl 2nd Strand Master Mix, pipet 6x gently to mix, cap & flick mix
Incubate on thermocycle e. 2nd Strand (16oC 60 m→25oC ∞)
Remove from thermocycle as soon as 60 min complete, let stand at RT
Vortex AMPure XP Beads to mix well
Add 90µl well-mixed beads to 50µl ds cDNA, cap, invert &/or flick to mix
Incubate 5 m @ RT, Condense beads into pellet on magnet ~3 m
§ Leave sample on magnet, DO NOT DISTURB condensed bead pellet
Remove & discard supernatant - DO NOT DISTURB BEAD PELLET! (DNDBP!)
Add 200µl 70% EtOH, incubate 30 s, remove & discard supernatant – DNDBP!
Add 200µl 70% EtOH, incubate 30 s, remove & discard supernatant– DNDBP!
Allow pellet to air dry @ RT on magnet, up to 15 m
Add 26.5µl Re-Suspension Buffer, pipet to gently elute sample
Incubate @ RT 2 m
Condense beads on magnet until solution clears, ~3 m
Transfer clear supernatant with ds cDNA into new, labeled tube
Nanodrop & calculate recovery
determine normalized ng amount to continue based on sample w/ lowest concentration
calculate µl volume/sample for normalized ng amount
µl/sample = ng / nanodrop reading (ng/µl)
label aliquots with sample name, date, ds cDNA, & freeze
Sample
Nanodrop (ng/µl)
Recovery (ng)
Recovery %
1ST SAFE STOPPING POINT!
ds cDNA samples can now be safely stored at -15oC to -80oC.
mRNA-Seq Page 3 of 7.
µl/sample
Date:______________________
PI/Project/Sample Name(s):__________________________________________
Double Stranded cDNA Library Preparation
End Repair:
Preparations:
§ Thaw 1 tube of End Repair Mix & Re-Suspension Buffer
§ Thaw ds cDNA samples (from 1st Safe Stop)
§ Briefly vortex & centrifuge once thawed
§ Bring AMPure XP Beads to RT(from 4oC) & vortex vigorously to thoroughly mix
§ Review & follow Protocol for Handling AMPure XP Beads
§ Preheat thermocycler to 30oC & pre-program the following:
i. 30C-30m: 30oC for 30m→25oC ∞
ii. 37C-30m: 37oC for 30 m→25oC ∞
iii. 30C-10m: 30oC for 10m→25oC ∞
§ calculate Re-Suspension Buffer volume to total 60µl
µl/sample for normalized ng amount
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µl Re-Suspension Buffer to total 60µl
Bring sample volume to a total of 60µl with Re-Suspension Buffer
Add 40µl End Repair Mix, pipet gently &/or cap & flick to mix
Thermocycle i. 30C-30m (30oC for 30m→25oC ∞)
Vortex AMPure XP Beads to mix well
Add 160µl well mixed Beads pipet gently to mix, cap, flick to mix
Incubate 5 m RT, Condense beads into pellet on magnet ~3 m
Leave sample on magnet, DO NOT DISTURB condensed bead pellet
Remove & discard supernatant - DO NOT DISTURB BEAD PELLET! (DNDBP!)
Add 200µl 70% EtOH, incubate 30 s, remove & discard supernatant – DNDBP!
Add 200µl 70% EtOH, incubate 30 s, remove & discard supernatant– DNDBP!
Allow pellet to air dry @ RT on magnet, up to 15 m
Add 18µl Re-Suspension Buffer, pipet to gently rinse ds cDNA from pellet
Cap, flick to mix & incubate 2 m RT
Condense beads on magnet until solution clears, ~3 m
Transfer clear supernatant to new tube as sample name, date & ds cDNA-ER
2nd SAFE STOPPING POINT!
End Repaired ds cDNA can be stored at -15oC to -80oC.
mRNA-Seq Page 4 of 7.
Date:______________________
PI/Project/Sample Name(s):__________________________________________
Double Stranded cDNA Library Preparation
Adenylate 3’Ends:
Preparations:
§ Thaw 1 tube of A-Tailing Mix, Re-Suspension Buffer & ds cDNA-ER samples
§ Pre-heat thermycycler to 37oC
o Add 12.5µl A-tailing Mix to samples, cap & flick to mix
o Incubate in thermocycler ii. 37C-30m (37oC for 30 m→25oC ∞)
o Remove from thermocycler when complete & proceed immediately to Adapter Ligation
Adapter Ligation:
Preparations:
§ Select 1 RNA Adapter Index per sample & record in table below
§ Thaw appropriate tubes of RNA Adapter Index & 1 tube Stop Ligase Mix
§ Thaw ligase control (if used)
§ Bring AMPure XP Beads to RT(from 4oC) & vortex vigorously to thoroughly mix
§ Immediately prior to use remove DNA Ligase Mix from -15oC to -25oC storage
o Add 2.5µl DNA Ligase Mix & 2.5µl Ligase Control (if used) or re-suspension buffer
§ Return DNA Ligase Mix to -15oC to -25oC storage immediately after use
o Add 2.5µl of selected RNA Adapter Index, pipet gently, cap & flick to mix
o Incubate on thermocycle iii. 30C-10m (30oC for 10m→25oC ∞)
o As soon as complete remove from thermocycler & Add 5µl Stop Ligase Mix, cap & flick
o Vortex AMPure XP Beads to evenly disperse & add 42µl, pipet gently, cap & flick to mix
o Incubate 5 m RT, Condense beads into pellet on magnet ~3 m
§ Leave sample on magnet, DO NOT DISTURB condensed bead pellet
o Remove & discard supernatant - DO NOT DISTURB BEAD PELLET! (DNDBP!)
o Add 200µl 70% EtOH, incubate 30 s, remove & discard supernatant – DNDBP!
o Add 200µl 70% EtOH, incubate 30 s, remove & discard supernatant– DNDBP!
o Allow pellet to air dry @ RT on magnet, up to 15 m
o Add 12µl Re-Suspension Buffer, pipet to gently rinse ds cDNA from pellet
o cap, flick to mix & incubate for @ RT for 2 m
o Condense beads on magnet until solution clears, ~3 m
o Transfer clear supernatant to new tube as sample name, date & ds cDNA-library
o Nanodrop, calculate recovery & volume to normalize amount/sample for PCR input
Sample Adapter
Nanodrop (ng/µl)
ds cDNA
Recovery
Vol to PCR
Index #
(ng)
%
(µl)
mRNA-Seq Page 5 of 7.
Date:______________________
PI/Project/Sample Name(s):__________________________________________
PCR Enrich Fragments:
Preparations:
§ Thaw 1 tube each of PCR Master Mix, PCR Primer Cocktail & sample
§ Centrifuge all briefly
§ Bring AMPure XP Beads to RT(from 4oC) & vortex vigorously to thoroughly mix
§ Pre-heat thermocycler to 100 oC &pre-program
PCR: 98oC for 30 s →
8-12 cycles of 98oC for 10 s→
60oC for 30 s→
72oC for 30 s→ then → 72oC for 5 m→10oC ∞
o Equalize volumes of normalized samples to 20µl each
Sample
Vol for normalized amt
Vol to total 20µl
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Add 5µl PCR Primer Cocktail
Add 25µl PCR Master Mix, cap & flick to mix, centrifuge briefly
Amplify on thermocycle PCR (as above)
Vortex AMPure XP Beads & add 40µl beads (0.8x sample volume)
pipet gently, cap, flick mix
Incubate 15 m RT, Condense beads into pellet on magnet ~3 m
§ Leave sample on magnet, DO NOT DISTURB condensed bead pellet
Remove & discard supernatant - DO NOT DISTURB BEAD PELLET! (DNDBP!)
Add 200µl 70% EtOH, incubate 30 s, remove & discard supernatant – DNDBP!
Add 200µl 70% EtOH, incubate 30 s, remove & discard supernatant– DNDBP!
Allow pellet to air dry @ RT on magnet, up to 15 m
Add 16.5µl Elution Buffer, pipet gently to rinse DNA from pellet
cap, flick to mix & incubate for @ RT for 2 m
Condense beads on magnet until solution clears, ~3 m
Transfer clear supernatant to new tube labeled with sample name, date & library
Nanodrop, calculate recovery
Nanodrop (ng/µl)
DNA (ng)
sample
4th Safe Stop Point: store samples between -15oC to -80oC.
mRNA-Seq Page 6 of 7.
Date:______________________
PI/Project/Sample Name(s):__________________________________________
Validate Libraries - Experion DNA chip:
o See Experion DNA protocol
o For single-read libraries final product should band at ~260bp.
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Quantify Libraries – Kappa Quant. qPCR:
Dilute samples 1:1000 with EB
Prepare the following recipe X total # of sample replicates & 6 standards +10%
- 12µl KAPA SYNBR FAST QPCR Master Mix
- 4µl diH2O
Set-up 96-well plate map as needed & aliquot 16µl above recipe/well to be used
Add 4µl of each diluted sample & standard in triplicate wells, pipet gently to mix
Run qPCR thermocycle protocol 95oC for 5 m→
35 cycles of: 95oC for 30 s→60oC for 45 s
Confirm samples in line with standards
Use absolute quantification to calculate library concentrations to load on flow cell
Cluster Flow Cell
o Dilute samples as needed based on Kappa Quantification & desired final flow cell
cluster densities
o See cBOT workflow protocol
Load flow cell on GA or HiSeq
mRNA-Seq Page 7 of 7.