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== RNAseIII-Fragmented Library Preparation == :'''RNA PREPARATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2ng/ul, 200pg/ul, 20pg/ul UHRR||||||||||0.5ul |- |ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul |- |10x RNaseIII Buffer||||||||||0.2ul |- |1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41 or STRT_V3_T30||||||||||0.1ul |- |dH20||||||||||0.25ul |- |Total||||||||||1.25ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1min'' :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA||||||||||1.25ul |- |RNase III||||||||||0.08ul |- |dH20||||||||||0.67ul |- |Total||||||||||2ul |- |} :::''Incubate @ 37C 10 min'' :::''Add 1.1ul dH2O :::''Incubate @ 65C 10 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||3.1ul |- |5x RT Buffer||||||||||0.5ul |- |1mM ATP||||||||||0.25ul |- |0.5U/ul PAP (1:10d in H2O)||||||||||0.25ul |- |} :::''Incubate @ 37C 5 min'' :::''Transfer to Ice'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||4.1ul |- |10mM dNTP Mix||||||||||1ul |- |1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41 or STRT_V3_T30||||||||||0.9ul |- |} :::''Incubate @ 65C 3 min'' :::''Transfer to Ice'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5xRT Buffer||||||||||1.5ul |- |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 ::#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 to 3ul :'''TSO''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |5x RT buffer [MMLV]||||||||||2ul |- |10mM dNTP mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |10uM STRT_V2_3 TSO||||||||||1ul |- |Betaine (5M)||||||||||2ul |- |RNase Inhibitor||||||||||0.25ul |- |Smarter MMLV||||||||||0.5ul |- |Total||||||||||10ul |- |} :::''Incubate @42C 10min'' :::''Incubate @70C 10min'' :'''USER''' ::''Add 1ul and incubate at 37C for 30 min'' :'''PhusionHF PCR (for T20V.ID)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||11ul |- |2x Buffer||||||||||25ul |- |10uM P1-STRT||||||||||1ul |- |10uM PCR_R_N2_id ||||||||||1ul |- |H2O||||||||||12ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 72C 20s||||||||||6x (1ng), 9x (100pg), 12x (10pg) |- |72C, 5min |- |} :'''KAPAHF PCR (STRT_V3_T30)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |cDNA||||||||||11ul |- |2x KAPA HiFi Hotstart ReadyMix||||||||||25ul |- |STRT_PCR (10mM)||||||||||1.0ul |- |dH2O||||||||||13ul |- |Total||||||||||50ul |- |} ::*98C 3min ::*12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg): :::*98C 15 sec :::*67C 20sec :::*72C 30sec ::*72C 5min
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