Blue:RNA-Seq Experiments:09182013: Difference between revisions
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== RNAseIII-Fragmented Library Preparation (1-Step) == | == RNAseIII-Fragmented Library Preparation (1-Step) == |
Revision as of 22:49, 17 September 2013
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_UHRR_ | UHRR/ERCC | 1ng - Unfragmented-PolyA | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Smarter Full-length totoRNAseq | Read 1 and N2 barcode read | ||
BL_UHRR_ | UHRR/ERCC | 100pg - Unfragmented-PolyA | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Smarter Full-length totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 10pg - Unfragmented-PolyA | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Smarter Full-length totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 1ng - Unfragmented | Smarter TSO (Kit) | SMART CDS (Kit) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 100pg - Unfragmented | Smarter TSO (Kit) | SMART CDS (Kit) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 10pg - Unfragmented | Smarter TSO (Kit) | SMART CDS (Kit) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 1ng - Unfragmented | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 100pg - Unfragmented | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 10pg - Unfragmented | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 1ng - RnaseIII Fragmented | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Smarter totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 100pg - RNaseIII Fragmented | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Smarter totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 10pg - RNaseIII Fragmented | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Smarter totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 1ng - RnaseIII Fragmented | STRT_V2_3 (In-house) | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 100pg - RNaseIII Fragmented | STRT_V2_3 (In-house) | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 10pg - RNaseIII Fragmented | STRT_V2_3 (In-house) | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 1ng - RNaseIII Fragmented - 1 step | TSO_r03 | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 100pg - RNaseIII Fragmented - 1 step | TSO_r03 | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 10pg - RNaseIII Fragmented - 1 step | TSO_r03 | T20V.id | N2_id | totoRNAseq |
Purpose
- Examine efficiency of Smarter protocol (unfragmented) compared to Poly-A tailing (unfragmented) and our fragmentation protocol using ERCC assay
- Examine efficiency of the modified TSO r03 (unable to be Poly-A tailed) in a single step totoRNAseq reaction using ERCC assay
Library Preparation (Unfragmented)
- RNA PREPARATION
Volume 2ng/ul, 200pg/ul, 20pg/ul UHRR 0.5ul ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul 3' CDS Primer IIA(12uM) or STRT_V3_T30(10uM) 1.0ul dNTP (10mM) 1ul dH2O 1.8ul Total 4.5ul
- Incubate @ 72C 3 min
- Transfer to ice
Volume 5xRT Buffer 2ul 100mM DTT 0.17ul Smarter II A TSO(12uM) or STRT_V2_3 TSO (10uM) 1ul Betaine 2ul RNase Inhibitor (40U/ul) 0.1ul Smarter MMLV (100U/ul) 0.23ul Total 10ul
- Incubate @ 42C 90 min
- Incubate @ 70C 10 min
- Beads Purification
- Add 15ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 10ul dH2O, incubate 2min, transfer to new tube
- PCR Amplification
Volume cDNA 10ul 2x KAPA HiFi Hotstart ReadyMix 25ul ISPCR (10mM) or STRT_PCR 1.0ul dH2O 14ul Total 50ul
- 98C 3min
- 12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg):
- 98C 15 sec
- 67C 20sec
- 72C 6min
- 72C 5min
Poly-A Unfragmented Library Preparation
- RNA PREPARATION
Volume 2ng/ul, 200pg/ul, 20pg/ul UHRR 0.5ul ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul 5x RT Buffer 0.5ul 1mM ATP 0.25ul 0.5U/ul PAP (1:10d in H2O) 0.25ul dH20 0.3ul Total 2.5ul
- Incubate @ 37C 5 min
- Transfer to Ice
- REVERSE TRANSCRIPTION
Volume PAP RNA 2.5ul 10mM dNTP Mix 1ul STRT_V3_T30 1ul dH2O 1.5ul
- Incubate @ 65C 3 min
- Transfer to Ice
Volume 5xRT Buffer 1.5ul 100mM DTT 0.17ul Betaine 2ul RNase Inhibitor (40U/ul) 0.1ul Smarter MMLV (200U/ul) 0.23ul Total 10ul
- Incubate @ 42C 90 min
- Beads Purification
- Add 15ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 10ul dH2O, incubate 2min, transfer to new tube
TSO
- PCR Amplification
Volume cDNA 10ul 2x KAPA HiFi Hotstart ReadyMix 25ul ISPCR primers (10mM) 1.0ul dH2O 14ul Total 50ul
- 98C 3min
- 12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg):
- 98C 15 sec
- 67C 20sec
- 72C 6min
- 72C 5min
RNAseIII-Fragmented Library Preparation
- RNA PREPARATION
Volume 2ng/ul, 200ng/ul, 20ng/ul UHRR 0.5ul ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul 10x RNaseIII Buffer 0.2ul 1uM T20V.id1 0.1ul dH20 0.25ul Total 1.25ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- RNA FRAGMENTATION
Volume RNA 1.25ul RNase III 0.08ul dH20 0.67ul Total 2ul
- Incubate @ 37C 10 min
- Add 1.1ul dH2O
- Incubate @ 65C 10 min
- 3' Tailing
Volume PNK_fRNA 3.1ul 5x RT Buffer 0.5ul 1mM ATP 0.25ul 0.5U/ul PAP (1:10d in H2O) 0.25ul
- Incubate @ 37C 5 min
- Transfer to Ice
- Reverse Transcription
Volume PAP RNA 4.1ul 10mM dNTP Mix 1ul 1uM T20V.ID 0.9ul
- Incubate @ 65C 3 min
- Transfer to Ice
Volume 5xRT Buffer 1.5ul 100mM DTT 0.17ul Betaine (5M) 2ul RNase Inhibitor (40U/ul) 0.1ul Smarter MMLV (200U/ul) 0.23ul Total 10ul
- Incubate @ 42C 30 min
- Beads Purification
- Add 25ul beads per well
- Allow to bind 15 min
- 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
- TSO (x2)
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
- USER
- Add 1ul and incubate at 37C for 30 min
- PCR (PhusionHF)
Volume TSO Reaction 11ul 2x Buffer 25ul 10uM P1-STRT (Smarter TSO) 1ul 10uM PCR_R_N2_id 1ul H2O 12ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 6x (1ng), 9x (100pg), 12x (10pg) 72C, 5min
RNAseIII-Fragmented Library Preparation (1-Step)
- RNA PREPARATION
Volume 2ng/ul, 200ng/ul, 20ng/ul UHRR 0.5ul ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul 10x RNaseIII Buffer 0.15ul 1uM T20V.id1 0.1ul Total 0.95ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- RNA FRAGMENTATION
Volume RNA 0.95ul RNase III 0.08ul dH20 0.47ul Total 1.5ul
- Incubate @ 37C 10 min
- Add 0.6ul dH2O
- Incubate @ 65C 10 min
- 3' Tailing
Volume fRNA 2.1ul 5x RT Buffer 0.5ul 1mM ATP 0.25ul 0.5U/ul PAP (1:10d in H2O) 0.25ul
- Incubate @ 37C 5 min
- Transfer to Ice
- Reverse Transcription
Volume PAP RNA 3.1ul 10mM dNTP Mix 1ul 1uM T20V.ID 0.9ul
- Incubate @ 65C 3 min
- Transfer to Ice
Volume 5xRT Buffer 1.5ul 100mM DTT 0.17ul Betaine (5M) 2ul TSO.r03 (10uM) 1ul RNase Inhibitor (40U/ul) 0.1ul Smarter MMLV (200U/ul) 0.23ul Total 10ul
- Incubate @42C 30 min
- Incubate @70C 10 min
- USER
- Add 1ul and incubate at 37C for 30 min
- PCR (PhusionHF)
Volume TSO Reaction 11ul 2x Buffer 25ul 10uM PB_PCR_F 1ul 10uM PCR_R_N2_id 1ul H2O 12ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 6x (1ng), 9x (100pg), 12x (10pg) 72C, 5min
Results of Amplification
- Ampure Beads Purify (0.8x)
- Resuspend in 10ul
- Run 1ul on gel