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== cDNA Preparation: Mg-based Fragmentation == :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2ng/ul or 0.2ng/ul or 0.02ng/ul ||||||||||0.5ul |- |1:10E4 or 1:10E5 or 1:10E6 ERCC ||||||||||0.2ul |- |5x RT buffer||||||||||1ul |- |dH2O||||||||||0.3ul |- |Total||||||||||2ul |- |} :::''Incubate @ 94C 2 min'' :::''Transfer to ice'' :'''End Repair''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||2ul |- |1mM ATP||||||||||0.4ul |- |100mM DTT||||||||||0.1ul |- |RNase Inhibitor||||||||||0.1ul |- |PNK||||||||||0.5ul |- |Total||||||||||3.1ul |- |} :::''Incubate @ 37C 30 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PNK_fRNA||||||||||3.1ul |- |1mM ATP||||||||||0.5ul |- |0.5U/ul Enzymatics PAP (1:10d in H2O)||||||||||0.5ul |- |Total|||||||||| 4.1 |- |} :::''Incubate @ 37C 5 min'' :::''Transfer to Ice'' :::''Add 1ul of 0.1uM T30VN.id'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||5ul |- |5xRT Buffer||||||||||1ul |- |10mM dNTP Mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |Betaine (5M)||||||||||2ul |- |Smarter MMLV (200U/ul)||||||||||0.25ul |- |dH2O||||||||||0.5ul |- |Total||||||||||10.1ul |- |} :::''Incubate @ 42C 30 min'' :::''For MARS-seq - continue to that section'' :'''Beads Purification''' ::#Add 15ul beads per well ::#Allow to bind 15 min ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry 10min or until cracking first occurs ::#Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin ~8min to 3ul
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