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== UMI-STRT-Seq Library Preparation == :'''TSO Reaction''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |5x First Strand buffer||||||||||1.4ul |- |10mM dNTP mix||||||||||0.7ul |- |100mM DTT||||||||||0.175ul |- |10uM C1-P1-RNA-TSO||||||||||0.7ul |- |RNase Inhibitor||||||||||0.175ul |- |SuperScriptII||||||||||0.7ul |- |dH2O||||||||||0.15ul |- |Total||||||||||7ul |- |} :::''Incubate @42C 20min'' :::''Incubate @70C 10min'' :'''PCR (Advantage)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||7ul |- |10x Advantage 2 Buffer||||||||||7ul |- |10uM C1-P1-PCR-2||||||||||2.8ul |- |dNTPs||||||||||2.8ul |- |Advantage 2 Polymerase||||||||||2.8ul |- |H2O||||||||||47.6ul |- |Total||||||||||70ul |- |} ::{| {{table}} |- |95C 60 sec|||||||||| |- |95C 20s -> 58C 4min -> 68C 6min||||||||||5x |- |95C 20s -> 64C 30s -> 68C 1min||||||||||10x (1ng); 13x (100pg); 16x (10pg) |- |72C, 10min |- |} *Run 7ul on gel: [[File:03052014_PCR1_Gel1_STRT.jpg|600px]] Results: Mg protocol does not seem to be compatible with Sten's latest STRT protocol (using RNA TSO) - possible carry over of Enzymatics PAP through beads purification. RNaseIII protocol (Affymetrics PAP) seems fine for all RNA amounts, but does show a ~600bp band. The NC for the RNaseIII STRT protocol does seem clean. Amplification levels for the RNaseIII protocol are lower than the totoRNAseq protocol. *Beads Purified (0.9x) twice and eluted in 10ul. Quantification using Qubit:
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