Blue:RNA-Seq Experiments:11072013
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RNA-Seq Library Information[edit]
Library ID | Samples | Input RNA | TSO Primer | Bar Code Primers | Bar Code Primer 2 | Type of Seq | Reads | Library Conc |
BL_ | UHRR/ERCC | 1ng | STRT_v2_3 | STRT_V3_T30 | Smart-Seq2 | |||
BL_ | UHRR/ERCC | 1ng | STRT_v2_3 | STRT_V3_T30 | Smart-Seq2 | |||
BL_ | UHRR/ERCC | 1ng - Poly(A) | STRT_v2_3 | STRT_V3_T30 | Smart-Seq2 | |||
BL_ | UHRR/ERCC | 1ng - Poly(A) | STRT_v2_3 | STRT_V3_T30 | Smart-Seq2 | |||
BL_ | UHRR/ERCC | 1ng - Poly(A) with dATP | STRT_v2_3 | STRT_V3_T30 | Smart-Seq2 | |||
BL_ | UHRR/ERCC | 1ng - Poly(A) with dATP | STRT_v2_3 | STRT_V3_T30 | Smart-Seq2 |
Purpose[edit]
- To test the effectiveness of Smart-Seq2 protocol for partially degraded samples and the potential rescuing effect of PAP
Smart-seq 2 Protocol[edit]
- RNA
Volume UHRR 2ng/ul 0.5ul ERCC 1:10E4 0.2ul dH2O 0.3ul 10uM T30 1ul dNTP (10mM) 1ul Total 3ul
- Incubate @ 72C 3 min
- Transfer to ice
- Reverse Transcription
Volume 5xRT Buffer 2ul 100mM DTT 0.25ul 100mM MgCl2 0.6ul Betaine (5M) 2ul TSO 1ul dH20 0.4ul RNase Inhibitor (40U/ul) 0.25ul Smarter MMLV (200U/ul) 0.5ul Total 10ul
- Incubate @ 42C 90 min
- Incubate @ 70C 10 min
- Beads Purification
- Add 25ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry until cracking first occurs
- Resuspend beads in PCR reaction:
- PCR (Advantage)
Volume 10x Advantage 2 PCR Buffer 5ul dNTP Mix (10mM) 2ul STRT Primer (10uM) 2ul 50X Advantage 2 Polymerase Mix 2ul H2O 39ul Total 50ul
95C 1 min 95C 15s -> 65C 30s -> 68C 6min 12x 72C, 10min
Smart-seq 2 Protocol with Poly(A) Tailing[edit]
- RNA
Volume UHRR 2ng/ul 0.5ul ERCC 1:10E4 0.2ul 5x ePAP Buffer 0.6ul dH2O 0.9ul Total 2.2ul
- Incubate @ 72C 3 min
- Transfer to ice
Volume 25mM MnCl2 0.3ul 1mM ATP or 1:1 ATP/dATP 0.25ul ePAP 2u/ul 0.25ul Total 3ul
- Incubate @ 37C 10 min
Volume 10uM T30 1ul dNTP (10mM) 1ul Total 5ul
- Reverse Transcription
Volume 5xRT Buffer 2ul 100mM DTT 0.25ul 100mM MgCl2 0.3ul Betaine (5M) 2ul RNase Inhibitor (40U/ul) 0.25ul Smarter MMLV (200U/ul) 0.5ul Total 10.3ul
- Incubate @ 42C 90 min
- Beads Purification
- Add 25ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry until cracking first occurs
- Resuspend beads in TSO reaction:
- TSO
Volume dH2O 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 (Advantage)
Volume 10x Advantage 2 PCR Buffer 5ul dNTP Mix (10mM) 2ul STRT Primer (10uM) 2ul 50X Advantage 2 Polymerase Mix 2ul H2O 29ul Total 50ul
95C 1 min 95C 15s -> 65C 30s -> 68C 6min 12x 72C, 10min
Results of Amplification[edit]
- Run 5ul on gel:
- Results: Can see that the overall efficiency is abnormally low. We have been having a problem with this and the Smart-Seq protocol lately for some unknown reason. Poly(A) tailing clearly increases the yields of cDNA.