Blue:RNA-Seq Experiments:totoRNAseq optimizations
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Experimental Overview
File:TotoRNAseq expanded optimization.jpg
Purpose
- Systematic analysis of different methods for addition of forward ILMN sequencing adapter to totoRNAseq libraries
- Comparison of Mg-based and RNaseIII based fragmentation methods
- Comparison of Enzymatics and Affymetrix PAP
RNA-Seq Library Information
Library ID | Samples | Condition | 5' Adaptor | 3' Adaptor | N2 PCR Rev Primer | Type of Seq |
BL_totoRNAseq_1ngUHRR_ | UHRR/ERCC | 2x1ng - 2min Mg Frag/Enzymatics PAP 5 min - STRT-Seq | TSO.r06 | T30VN_id1 | N2.id1-2 | totoRNAseq |
UHRR/ERCC | 2x100pg - 2min Mg Frag/Enzymatics PAP 5 min - STRT-Seq | TSO.r06 | T30VN_id1 | N2.id3-4 | totoRNAseq | |
UHRR/ERCC | 2xNTC - 2min Mg Frag/Enzymatics PAP 5 min - STRT-Seq | TSO.r06 | T30VN_id1 | N2.id5-6 | totoRNAseq | |
UHRR/ERCC | 10x10pg+2xNTC - 2min Mg Frag/Enzymatics PAP 5 min - STRT-Seq | TSO.r06 | T30VN_id1-12 | N2.id7 | totoRNAseq | |
UHRR/ERCC | 2x1ng - 0.1U RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq | TSO.r06 | T30VN_id1 | N2.id8-9 | totoRNAseq | |
UHRR/ERCC | 2x100pg - 0.1U RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq | TSO.r06 | T30VN_id1 | N2.id10-11 | totoRNAseq | |
UHRR/ERCC | 2xNTC - 0.1U RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq | TSO.r06 | T30VN_id1 | N2.id12-13 | totoRNAseq | |
UHRR/ERCC | 10x10pg+2xNTC - 0.05U RNase3 Frag/Affymetrix PAP 4 min - STRT-Seq | TSO.r06 | T30VN_id1-12 | N2.id14 | totoRNAseq | |
UHRR/ERCC | 2x1ng - 2min Mg Frag/Enzymatics PAP 5 min - Quartz-Seq | STRT_T24 | T30VN_id1 | N2.id15-16 | totoRNAseq/Quartz-Seq | |
UHRR/ERCC | 2x100pg - 2min Mg Frag/Enzymatics PAP 5 min - Quartz-Seq | STRT_T24 | T30VN_id1 | N2.id17-18 | totoRNAseq/Quartz-Seq | |
UHRR/ERCC | 2xNTC - 2min Mg Frag/Enzymatics PAP 5 min - Quartz-Seq | STRT_T24 | T30VN_id1 | N2.id19-20 | totoRNAseq/Quartz-Seq | |
UHRR/ERCC | 10x10pg>2xNTC - 2min Mg Frag/Enzymatics PAP 5 min - Quartz-Seq | STRT_T24 | T30VN_id1-12 | N2.id21 | totoRNAseq/Quartz-Seq | |
UHRR/ERCC | 2x1ng - 0.1U RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq | STRT_T24 | T30VN_id1 | N2.id22-23 | totoRNAseq/Quartz-Seq | |
UHRR/ERCC | 2x100pg - 0.1U RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq | STRT_T24 | T30VN_id1 | N2.id24-25 | totoRNAseq/Quartz-Seq | |
UHRR/ERCC | 2xNTC - 0.1U RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq | STRT_T24 | T30VN_id1 | N2.id26-27 | totoRNAseq/Quartz-Seq | |
UHRR/ERCC | 10x10pg>2xNTC - 0.05U RNase3 Frag/Affymetrix PAP 5 min - Quartz-Seq | STRT_T24 | T30VN_id1-12 | N2.id28 | totoRNAseq/Quartz-Seq | |
UHRR/ERCC | 2x1ng - 2min Mg Frag/Enzymatics PAP 5 min - Cel-Seq | STRT_ADPT | T30VN_id1/RL_Add.T7 | N2.id29-30 | totoRNAseq/Cel-Seq | |
UHRR/ERCC | 2x100pg - 2min Mg Frag/Enzymatics PAP 5 min - Cel-Seq | STRT_ADPT | T30VN_id1/RL_Add.T7 | N2.id31-32 | totoRNAseq/Cel-Seq | |
UHRR/ERCC | 2xNTC - 2min Mg Frag/Enzymatics PAP 5 min - Cel-Seq | STRT_ADPT | T30VN_id1/RL_Add.T7 | N2.id33-34 | totoRNAseq/Cel-Seq | |
UHRR/ERCC | 10x10pg>2xNTC - 2min Mg Frag/Enzymatics PAP 5 min - Cel-Seq | STRT_ADPT | T30VN_id1-12/RL_Add.T7 | N2.id35 | totoRNAseq/Cel-Seq | |
UHRR/ERCC | 2x1ng - 0.1U RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq | STRT_ADPT | T30VN_id1/RL_Add.T7 | N2.id36-37 | totoRNAseq/Cel-Seq | |
UHRR/ERCC | 2x100pg - 0.1U RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq | STRT_ADPT | T30VN_id1/RL_Add.T7 | N2.id38-39 | totoRNAseq/Cel-Seq | |
UHRR/ERCC | 2xNTC - 0.1U RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq | STRT_ADPT | T30VN_id1/RL_Add.T7 | N2.id40-41 | totoRNAseq/Cel-Seq | |
UHRR/ERCC | 10x10pg>2xNTC - 0.05U RNase3 Frag/Affymetrix PAP 4 min - Cel-Seq | STRT_ADPT | T30VN_id1-12/RL_Add.T7 | N2.id42 | totoRNAseq/Cel-Seq | |
UHRR/ERCC | 2x1ng - 2min Mg Frag/Enzymatics PAP 5 min - Nugen | STRT_N8 | T30VN_id1 | N2.id43-44 | totoRNAseq/Nugen | |
UHRR/ERCC | 2x100pg - 2min Mg Frag/Enzymatics PAP 5 min - Nugen | STRT_N8 | T30VN_id1 | N2.id45-46 | totoRNAseq/Nugen | |
UHRR/ERCC | 2xNTC - 2min Mg Frag/Enzymatics PAP 5 min - Nugen | STRT_N8 | T30VN_id1 | N2.id47-48 | totoRNAseq/Nugen | |
UHRR/ERCC | 10x10pg>2xNTC - 2min Mg Frag/Enzymatics PAP 5 min - Nugen | STRT_N8 | T30VN_id1-12 | N2.id49 | totoRNAseq/Nugen | |
UHRR/ERCC | 2x1ng - 0.1U RNase3 Frag/Affymetrix PAP 10 min - Nugen | STRT_N8 | T30VN_id1 | N2.id50-51 | totoRNAseq/Nugen | |
UHRR/ERCC | 2x100pg - 0.1U RNase3 Frag/Affymetrix PAP 10 min - Nugen | STRT_N8 | T30VN_id1 | N2.id52-53 | totoRNAseq/Nugen | |
UHRR/ERCC | 2xNTC - 0.1U RNase3 Frag/Affymetrix PAP 10 min - Nugen | STRT_N8 | T30VN_id1 | N2.id54-55 | totoRNAseq/Nugen | |
UHRR/ERCC | 10x10pg>2xNTC - 0.05U RNase3 Frag/Affymetrix PAP 4 min - Nugen | STRT_N8 | T30VN_id1-12 | N2.id56 | totoRNAseq/Nugen |
Primer sequences [1]
cDNA Preparation: RNAseIII-Based Fragmentation
- RNA
Volume 2ng/ul 0.5ul 1:104 ERCC 0.2ul 0.1uM T20VN 0.1ul dH2O 0.2ul Total 1ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1 min
Volume 10x RNaseIII buffer 0.2ul RNase III 0.1ul dH20 0.7ul Total 2ul
- Incubate @ 37C 1 min
- 3' Tailing
Volume fRNA 2ul 5x RT buffer 0.5ul 1mM ATP 0.5ul 0.5U/ul Enzymatics PAP (1:10d in H2O) 0.5ul + 0.3ul dH2O or 600U/ul Affymetrix PAP 0.2ul + 0.6ul dH2O 40U/ul Rnase Inhibitor 0.2ul Total 4ul
- Incubate @ 37C 5 min (Enzymatics) or 4-10 min (Affymetrix)
- Add 1ul of 0.1uM T20VN.id
- Reverse Transcription
Volume PAP RNA 5ul 5xRT Buffer 1.5ul 10mM dNTP Mix 1ul 100mM DTT 0.25ul Betaine (5M) 2ul Smarter MMLV (200U/ul) 0.25ul Total 10ul
- Incubate @ 42C 30 min
- Beads Purification
- Add 15ul beads per well
- Allow to bind 15 min, combine wells
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin ~8min to 3ul
cDNA Preparation: Mg-based Fragmentation
- RNA FRAGMENTATION
Volume 2ng/ul UHRR RNA 0.5ul 1:104 ERCC 0.2ul 5x RT buffer 1ul dH2O 0.3ul Total 2ul
- Incubate @ 94C 2 min
- Transfer to ice
- End Repair
Volume fRNA 2ul 1mM ATP 0.4ul 100mM DTT 0.1ul RNase Inhibitor 0.1ul PNK 0.5ul Total 3.1ul
- Incubate @ 37C 30 min
- 3' Tailing
Volume PNK_fRNA 3.1ul 1mM ATP 0.5ul 0.5U/ul Enzymatics PAP (1:10d in H2O) 0.5ul or 600U/ul Affymetrix PAP 0.2ul + 0.3ul dH2O
- Incubate @ 37C 5 min (Enzymatics) or 10 min (Affymetrix)
- Transfer to Ice
- Add 1ul of 0.1uM T20VN.id
- Reverse Transcription
Volume PAP RNA 5ul 5xRT Buffer 1ul 10mM dNTP Mix 1ul 100mM DTT 0.25ul Betaine (5M) 2ul Smarter MMLV (200U/ul) 0.25ul dH2O 0.5ul Total 10ul
- Incubate @ 42C 30 min
- Beads Purification
- Add 15ul beads per well
- Allow to bind 15 min, combine wells
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin ~8min to 3ul
STRT-Seq Library Preparation
- TSO Reaction
Volume Purified cDNA 3ul 5x RT buffer [MMLV] 2ul 10mM dNTP mix 1ul 100mM DTT 0.25ul 10uM TSO 1ul Betaine (5M) 2ul RNase Inhibitor 0.25ul Smarter MMLV 0.5ul Total 10ul
- Incubate @42C 10min
- Incubate @70C 10min
- PCR (PhusionHF)
Volume TSO Reaction 10ul 2x Buffer 25ul 10uM P1-STRT 1ul 10uM PCR_R_N2_id 1ul H2O 13ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 9x (1ng) 72C, 5min
- Run 5ul on gel:
Results:
Quartz-Seq Library Preparation
Primer Digestion
Volume Purified cDNA 3ul 10x PCR Buffer 0.4ul 10x Exo I buffer 0.2ul 100mM DTT 0.2ul Exonuclease I 0.6ul dH2O 1.6ul Total 6ul
- Incubate @37C 30min
- Incubate @80C 20min
Poly-A Tailing
Volume ExoI RXN 6ul 10x PCR Buffer 0.5ul 100mM dATP 0.15ul RNaseH 0.12ul TdT enzyme 0.42ul dH2O 3.81ul Total 11ul
- Incubate @37C 50 sec
- Incubate @65C 10min
Second Strand Synthesis
Volume Poly-A Tailed Sample 11ul 2x PhusionHF Flex 25ul 10uM Smarter Tagging primer 0.32ul dH2O 20.68ul Total 57ul
- Incubate @98C 30sec
- Incubate @40C 1min
- Incubate @68C 5min
PCR Amplification
Volume 2nd Strand Sample 57ul 2x PhusionHF Flex 25ul 100uM P1-STRT 1ul 100uM PCR_R_N2.id 1ul dH2O 23ul Total 107ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 9x (1ng) or 12x (100pg) 72C, 5min
Cel-Seq Library Preparation
Second Strand Synthesis
Volume Purified cDNA 3ul FS Buffer 0.2ul SS Buffer 1ul 10mM dNTP 0.8ul DNA Polymerase 0.2ul RNaseH 0.1ul dH2O 4.7ul Total 10ul
- Incubate @16C 2 hours
T7 Primer Addition
Volume Second Strand Sample 10ul KAPA qPCR MM 25ul RL.Add.T7 Primer (10uM) 0.5ul dH2O 14.5ul Total 50ul
- Incubate @94C 2min
- Incubate @60C 2min
- Incubate @72C 5min
Beads Purification
- Add 50ul beads per well
- Allow to bind 15 min, combine wells
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 6.4ul dH2O, incubate 2min, transfer to new tube
IVT
Volume Purified T7-added Sample 6.4ul ATP 1.6ul GTP 1.6ul CTP 1.6ul UTP 1.6ul 10x T7 buffer 1.6ul T7 Enzyme 1.6ul Total 16ul
- Incubate @37C (lid 70C) 13 hours
- Incubate @4C
Quantification of aRNA
- Determine concentration of amplified RNA using Qubit
- Dilute 5-10ng RNA into 16ul dH2O
Phosphatase Treatment
Volume aRNA 16ul 10x Phosphatase buffer 2ul Anarctic phosphatase 1ul RNase Inhibitor 1ul Total 20ul
- Incubate @37C 30min
- Incubate @65C 5min
- Incubate @4C Indefinitely
PNK Treatment
Volume Phos Treated aRNA 20ul dH2O 17ul 10x phosphatase buffer 5ul ATP (10mM, from Illumina kit) 5ul RNase Inhibitor 1ul PNK 2ul Total 50ul
- Incubate @37C 60min
RNA Cleanup: (Zymo RNA Clean and Concentrator-5)
- Add 100ul RNA Binding Buffer and mix well
- Add 150ul 95-100% Ethanol and mix well
- Transfer to Zymo-Spin IC Column and centrifuge 1min
- Add 400ul RNA Prep Buffer to column and centrifuge 1min
- Add 800ul RNA Wash Buffer to column and centrifuge 1min
- Add 400ul RNA Wash Buffer to column and centrifuge 2min
- Transfer column to 1.5ml tube, add 6ul dH2O, 1min RT, centrifuge 1min
- Speedvac to 5ul
3'Adaptor Ligation
Volume Phos/PNK aRNA 5ul 3' Smarter Adaptor RA3 (1:5 dilution?) 1ul
- Incubate @70C 2min
- transfer to ice
Volume aRNA/adaptor 6ul 5x HM Ligation Buffer (HML, Illumina kit) 2ul RNase Inhibitor 1ul T4 RNA Ligase 2, truncated 1ul
- Incubate @28C 1 hour (unheated lid)
- Still in thermocycler: add 1ul stop solution (STP)
- Pipette entire volume 6-8 times to mix
- Incubate @28C 15min
- Transfer to ice
- Add 3ul dH2O
Reverse Transcription
Volume Adaptor-ligated RNA 6ul (store remaining at -80C) RNA Smarter RT Primer (RTP) 1ul
- Incubate @70C 2min
- Immediately transfer to ice
Volume RNA/primer 7ul 10x RT Buffer 1ul 10mM dNTP 0.5ul 100mM DTT 0.5ul RNase Inhibitor 0.5ul SuperScriptIII 0.5ul Total 10ul
- Incubate @25C 5min
- Incubate @50C 20min
- Incubate @70C 10min
PCR Amplification
Volume RT Reaction 10ul 2x PhusionHF Flex Buffer 25ul 10uM P1-STRT 1ul 10uM PCR_R_N2_id 1ul H2O 13ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 9x (10ng) 72C, 5min
Nugen Ovation Library Preparation
Second Strand Synthesis
Volume cDNA 3ul 2x PhusionHF Flex 5ul 10uM Smarter random primer 0.3ul RNaseH 0.1ul dH2O 1.6ul Total 10ul
- Incubate @37C 20min
- Incubate @98C 30sec
- Incubate @25C 30min
- Incubate @70C 10min
PCR Amplification
Volume 2nd Strand Sample 10ul 2x PhusionHF Flex 50ul 100uM P1-STRT 1ul 100uM PCR_R_N2.id 1ul dH2O 38ul Total 100ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 9x (1ng) or 12x (100pg) 72C, 5min