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Blue:RNA-Seq Experiments:totoRNAseq optimizations
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== MARS-Seq Library Preparation == '''Exonuclease Digestion''' :::''Add 1ul Exonuclease I (1:10 diluted in 1x exonuclease buffer)to each RT reaction'' :::''Incubate at 37C for 30min'' :::''Incubate at 80C for 10min'' :''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 17ul 10mM Tris-HCl pH7.5, incubate 2min, transfer to new tube '''Second Strand Synthesis''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |cDNA||||||||||17ul |- |10x NEB second strand synthesis buffer||||||||||2ul |- |Second strand synthesis enzyme||||||||||1ul |- |Total||||||||||20ul |- |} :::''Incubate @ 16C for 2.5 hours'' '''IVT''' ::#Add 28ul 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: ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x Reaction Buffer (T7 high yield NEB)||||||||||2ul |- |UTP||||||||||2ul |- |ATP||||||||||2ul |- |CTP||||||||||2ul |- |GTP||||||||||2ul |- |T7 polymerase||||||||||2ul |- |dH2O||||||||||8ul |- |Total||||||||||20ul |- |} :::''Incubate 16 hours (overnight) @ 37C''' :::''Add 1ul Turbo DNase I and incubate @ 37C 15min :''Beads Purification:'' ::#Add 24ul 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 10ul dH20, transfer to new tube, vacuspin to 3ul '''Adaptor Ligation''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |aRNA||||||||||3ul |- |100uM Lig_NNNX4_idx||||||||||1ul |- |} :::''Incubate @ 70C for 3min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |aRNA/adaptor||||||||||4ul |- |10x T4 Ligase buffer||||||||||1ul |- |DMSO||||||||||0.95ul |- |10mM ATP||||||||||1ul |- |PEG8000||||||||||2ul |- |T4 RNA Ligase I(10U/ul)||||||||||1ul |- |dH2O||||||||||0ul |- |Total||||||||||10ul |- |} :::''Incubate at 22C for 2hrs'' '''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Ligated RNA||||||||||8ul |- |10uM MARS_RT_2||||||||||1ul |- |10mM dNTP||||||||||1ul |- |Total||||||||||10ul |- |} :::''Incubate @ 65C 5min'' :::''Incubate on ice 1min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA/Primer||||||||||10ul |- |10x RT Buffer||||||||||2ul |- |25mM MgCl2||||||||||0.8ul |- |100mM DTT||||||||||2ul |- |RNase Inhibitor (40U/ul)||||||||||1ul |- |SSIII (200U/ul)||||||||||1ul |- |dH2O||||||||||3.2ul |- |Total||||||||||10ul |- |} :::''Incubate @42C 10min'' :::''Incubate @50C 50min'' :::''Incubate @85C 5min'' '''Beads Purify''' ::#Add 30ul 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 10ul dH20, transfer to new tube '''PCR Amplification''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |cDNA||||||||||10ul |- |SybrGreen KAPA 2x MM||||||||||25ul |- |10uM P5_Rd1_PCR_F||||||||||2.5ul |- |10uM P7_Rd2_PCR_R||||||||||2.5ul |- |dH2O|||||||||| |- |} ::{| {{table}} |- |95C 30 sec|||||||||| |- |95C 15s -> 60C 20s -> 72C 30s||||||||||12x |- |72C, 10min |- |}
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