Blue:RNA-Seq Protocols:Smart-seq2
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RNA-Seq Library Information[edit]
Input RNA | ERCC Dilution | T30 Primer | TSO Primer | PCR Primer | PCR Cycles | Protocol |
1ng UHRR | 1:10e5 | Smart2_T30VN | Smart2_TSO | IS PCR Primer | 15 | Smart-Seq2 |
100pg UHRR | 1:10e6 | Smart2_T30VN | Smart2_TSO | IS PCR Primer | 18 | Smart-Seq2 |
10pg UHRR | 1:10e7 | Smart2_T30VN | Smart2_TSO | IS PCR Primer | 21 | Smart-Seq2 |
Notes[edit]
- Smart-Seq2 protocol (Picelli et al., 2014)
- Using Lysis and PCR cycling adapted from the C1 protocol
- Using modified RT/TSO conditions adapted from totoRNAseq protocol (to incorporate MMLV vs Superscript)
Smart-seq 2 Protocol[edit]
- Lysis
Volume UHRR(or single cell in Clontech Dil buffer)/ERCC 2ul 10uM T30 1ul dNTP (10mM) 1ul Total 4ul
- Incubate @ 72C 3 min
- Incubate @ 4C 10 min
- Incubate @ 25C 1 min
- Incubate @ 4C hold
- Reverse Transcription
Volume 5xRT Buffer 2ul 100mM DTT 0.25ul Betaine (5M) 2ul 10uM TSO 1ul RNase Inhibitor (40U/ul) 0.25ul Smarter MMLV (200U/ul) 0.5ul Total 10ul
- Incubate @ 42C 90 min
- Incubate @ 70C 10 min
- PCR
Volume 2x PhusionHF Buffer 25ul IS Primer (10uM) 2ul H2O 13ul Total 50ul
98C 30 sec 98C 20s -> 58C 4min -> 68C 6min 5x 98C 20s -> 64C 30s -> 68C 6min 10x (1ng); 13x (100pg); 16x (10pg) 72C, 10min
Results of Amplification[edit]
- Run 5ul on gel: