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

Primer List


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:


Final Library Preparation[edit]