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== RNA-Seq Library Information == {| {{table}} border | align="center" style="background:#f0f0f0;"|'''Input RNA''' | align="center" style="background:#f0f0f0;"|'''ERCC Dilution''' | align="center" style="background:#f0f0f0;"|'''T30 Primer''' | align="center" style="background:#f0f0f0;"|'''TSO Primer''' | align="center" style="background:#f0f0f0;"|'''PCR Primer''' | align="center" style="background:#f0f0f0;"|'''PCR Cycles''' | align="center" style="background:#f0f0f0;"|'''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 |- |} [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List Primer List] == Notes == * 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 == :'''Lysis''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2x PhusionHF Buffer||||||||||25ul |- |IS Primer (10uM)||||||||||2ul |- |H2O||||||||||13ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |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 == *Run 5ul on gel: == Final Library Preparation ==
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