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 | Input RNA | TSO Primer | Bar Code Primers | Bar Code Primer 2 | Type of Seq | Reads | Library Conc |
BL_totoRNAseq_1ngUHRR_ | UHRR/ERCC | 4x1ng - Mg Frag/Enzymatics PAP 5 min - STRT-Seq | TSO.r06 | T20VN_id1-4 | N2.id1 | totoRNAseq | ||
UHRR/ERCC | 4x100pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq | TSO.r06 | T20VN_id5-8 | N2.id2 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq | TSO.r06 | T20VN_id9-18 | N2.id3 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq | TSO.r06 | T20VN_id1-4 | N2.id4 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq | TSO.r06 | T20VN_id5-8 | N2.id5 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq | TSO.r06 | T20VN_id9-18 | N2.id6 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq | xxx | T20VN_id1-4 | N2.id7 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq | xxx | T20VN_id5-8 | N2.id8 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq | xxx | T20VN_id9-18 | N2.id9 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq | xxx | T20VN_id1-4 | N2.id10 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq | xxx | T20VN_id5-8 | N2.id11 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq | xxx | T20VN_id9-18 | N2.id12 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Enzymatics PAP 5 min - Cel-Seq | xxx | T20VN_id1-4 | N2.id13 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq | xxx | T20VN_id5-8 | N2.id14 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq | xxx | T20VN_id9-18 | N2.id15 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq | xxx | T20VN_id1-4 | N2.id16 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq | xxx | T20VN_id5-8 | N2.id17 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq | xxx | T20VN_id9-18 | N2.id18 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Enzymatics PAP 5 min - Nugen | xxx | T20VN_id1-4 | N2.id19 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - Mg Frag/Enzymatics PAP 5 min - Nugen | xxx | T20VN_id5-8 | N2.id20 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - Mg Frag/Enzymatics PAP 5 min - Nugen | xxx | T20VN_id9-18 | N2.id21 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Nugen | xxx | T20VN_id1-4 | N2.id22 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen | xxx | T20VN_id5-8 | N2.id23 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen | xxx | T20VN_id9-18 | N2.id24 | totoRNAseq |
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 10uM P1-STRT 1ul 10uM 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 Purified cDNA 3ul FS Buffer 0.2ul