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== cDNA Preparation: RNAseIII-Based Fragmentation == :'''RNA''' ::{| {{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 |- |xuM PolyT primer (see below)||||||||||0.1ul |- |dH2O||||||||||0.2ul |- |Total||||||||||1ul |- |} Note: NTC with 1:10E6 ERCC and no RNA - treated as 10pg... :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1 min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x RNaseIII buffer||||||||||0.2ul |- |RNase III||||||||||0.1ul |- |dH20||||||||||0.7ul |- |Total||||||||||2ul |- |} :::''Incubate @ 37C 1 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||2ul |- |5x RT buffer||||||||||0.5ul |- |1mM ATP||||||||||0.5ul |- |600U/ul Affymetrix PAP||||||||||0.2ul |- |40U/ul Rnase Inhibitor||||||||||0.2ul |- |dH2O||||||||||0.6ul |- |Total||||||||||4ul |- |} :::''Incubate @ 37C 4-10 min'' :::''Add 1ul of 0.1uM T20VN.id [totoRNAseq]'' :::''Add 1ul of 12uM C1-P1-T31 [UMI-STRT-Seq]'' :::''Add 1ul of 10uM Smart2-T30VN [Smart-seq2]'' :::''Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]'' :::''Add 1ul of 2uM MARS_T20N_id [MARS-Seq]'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||5ul |- |5xRT Buffer||||||||||1.5ul |- |10mM dNTP Mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |Betaine (5M)||||||||||2ul |- |Smarter MMLV (200U/ul)||||||||||0.25ul |- |Total||||||||||10ul |- |} :::''Incubate @ 42C 60 min'' :::''For MARS-seq - continue to that section'' :'''Beads Purification''' ::#Add 25ul 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 dH20; incubate 2min RT; transfer to new tube and vacuum spin to ~3ul (~8min)
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