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== RNAseIII-Fragmented Library Preparation == :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |UHRR 200pg/ul ||||||||||0.5ul |- |ERCC 1:10E5||||||||||0.2ul |- |5x Ambion PAP buffer||||||||||0.08ul |- |0.1uM T20V.id2||||||||||0.1ul |- |25mM MnCl2||||||||||0.08ul |- |dH20||||||||||0.29ul |- |Total||||||||||1.25ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1min'' :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA||||||||||1.25ul |- |RNase III||||||||||0.08ul or 0.04ul or 0.01ul |- |dH20||||||||||0.67ul or 0.71ul or 0.74ul |- |Total||||||||||2ul |- |} :::''Incubate @ 37C 5 min'' :::''Incubate @ 65C 10 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||2ul |- |5x Ambion PAP Buffer||||||||||0.72ul |- |25mM MnCl2||||||||||0.32ul |- |ATP (or mix)||||||||||0.25ul |- |0.4U/ul PAP||||||||||0.2ul |- |dH2O||||||||||0.51ul |- |} :::''Incubate @ 37C 10 min'' :::''Transfer to Ice'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||4ul |- |10mM dNTP Mix||||||||||1ul |- |0.2uM T20V.ID ||||||||||0.5ul |- |} :::''Incubate @ 65C 10 min'' :::''Transfer to Ice'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5xRT Buffer||||||||||2ul |- |100mM DTT||||||||||0.17ul |- |Betaine (5M)||||||||||2ul |- |RNase Inhibitor (40U/ul)||||||||||0.1ul |- |Smarter MMLV (200U/ul)||||||||||0.23ul |- |Total||||||||||10ul |- |} :::''Incubate @ 42C 30 min'' :'''Beads Purification''' ::#Add 15ul beads per well ::#Allow to bind 15 min - combine sets ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry 10min or until cracking first occurs ::#Resuspend beads in 10ul dH2O, incubate 2min, transfer to new tube - vacuspin 8 min to bring to 3ul :'''TSO''' ::{| {{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 10min'' :::''Incubate @70C 10min'' :'''PCR (PhusionHF)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||10ul |- |2x Buffer||||||||||25ul |- |10uM P1-STRT||||||||||1ul |- |10uM PCR_R_N2_id||||||||||1ul |- |H2O||||||||||13ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 72C 20s||||||||||12x |- |72C, 5min |- |}
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