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== Smart-Seq2 Library Preparation == :'''TSO Reaction''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |5x First Strand buffer||||||||||2ul |- |10mM dNTP mix||||||||||1ul |- |100mM DTT||||||||||0.5ul |- |100uM Smart2-TSO||||||||||0.1ul |- |MgCl2 (1M)||||||||||0.06ul |- |Betaine (5M)||||||||||2ul |- |RNase Inhibitor||||||||||0.25ul |- |SuperScriptII||||||||||0.5ul |- |dH2O||||||||||0.59ul |- |Total||||||||||10ul |- |} :::''Incubate @42C 20min'' :::''Incubate @70C 10min'' :::''Incubate @10C hold'' :'''PCR (KAPA-HiFi)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||10ul |- |2x KAPA HiFi Hotstart ReadyMix||||||||||12.5ul |- |IS PCR Primer (10uM)||||||||||0.25ul |- |H2O||||||||||2.25ul |- |Total||||||||||25ul |- |} ::{| {{table}} |- |98C 3min|||||||||| |- |98C 20s -> 67C 15s -> 72C 1min||||||||||15x (1ng); 18x (100pg); 21x (10pg) |- |72C, 5min |- |} [[File:03052014_PCR1_Gel1_Smart2.jpg|400px]] Results: Can see that the PCR did not work. Maybe the conditions are sub-optimal for fragmented RNA. Could try similar conditions as the STRT-seq or totoRNAseq... '''Repeat Experiment''' :'''TSO Reaction''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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 20min'' :::''Incubate @70C 10min'' :'''PCR (PhusionHF)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||10ul |- |2x Buffer||||||||||25ul |- |IS PCR Primer (10uM)||||||||||2ul |- |H2O||||||||||13ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 64C 20s -> 72C 20s||||||||||9x (1ng); 12x (100pg); 15x (10pg) |- |72C, 5min |- |} *Run 5ul on gel: [[File:03052014_PCR1_Gel1_Smart2_2.jpg|400px]] Results: Appears that the Smart-seq2 protocol is also incompatible with the Mg RNA fragmentation protocol (likely due to the enzymatics PAP). For RNaseIII fragmented RNA, the efficiency looks to be too low to amplify 10pg libraries... therefore the Smart-seq2 adaptation may not work well enough to continue. cDNA quantification is needed to determine which samples can be used for Nextera library preparation.
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