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== RNA-Seq Library Information == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Input RNA''' | align="center" style="background:#f0f0f0;"|'''TSO Primer''' | align="center" style="background:#f0f0f0;"|'''Bar Code Primers''' | align="center" style="background:#f0f0f0;"|'''Bar Code Primer 2''' | align="center" style="background:#f0f0f0;"|'''Type of Seq''' | align="center" style="background:#f0f0f0;"|'''Reads''' | align="center" style="background:#f0f0f0;"|'''Library Conc''' |- | BL_||UHRR/ERCC||100pg - RNase3(.08) 5 min/PAP 10min||TSO.r06||T20V.id2||N2.id10||totoRNAseq|||| |- | BL_||UHRR/ERCC||100pg - RNase3(.08) 5 min/PAP 10min/1:0.01 ATP||TSO.r06||T20V.id2||N2.id11||totoRNAseq|||| |- | BL_||UHRR/ERCC||100pg - RNase3(.08) 5 min/PAP 10min/1:0.05 ATP||TSO.r06||T20V.id2||N2.id12||totoRNAseq|||| |- | BL_||UHRR/ERCC||100pg - RNase3(.08) 5 min/PAP 10min/1:0.1 ATP||TSO.r06||T20V.id2||N2.id13||totoRNAseq|||| |- | BL_||UHRR/ERCC||100pg - RNase3(.04) 5 min/PAP 10min/1:0.05 ATP||TSO.r06||T20V.id2||N2.id14||totoRNAseq|||| |- | BL_||UHRR/ERCC||100pg - RNase3(.01) 5 min/PAP 10min/1:0.05 ATP||TSO.r06||T20V.id2||N2.id15||totoRNAseq|||| |- | BL_||UHRR/ERCC||100pg - no RNase3 5 min/PAP 10min/1:0.05 ATP||TSO.r06||T20V.id2||N2.id16||totoRNAseq|||| |- |} Note: ATP Condtions: #1mM ATP/0mM 3'dATP #1mM ATP/0.01mM 3'dATP #1mM ATP/0.05mM 3'dATP #1mM ATP/0.1mM 3'dATP == Purpose == *Continuation of tests from 10-23-2013 *To try and reduce the number of PolyA reads in the totoRNAseq library that might arise from overfragmentation or overactive PAP **Test [RNaseIII] on fragmentation and resultant post-PCR amplification **Test termination of PAP activity to limit poly-A tail length by adding Cordycepin-ATP or 3'-ATP at different ratios to regular ATP **Include PAP inactivation - heating == 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 |- |} == Results of Amplification == *Run 5ul on gel: [[File:10292013_PCR1_gel1.jpg]] Note: Numbers refer to: #id10 [RNaseIII (0.08U) 5min]/[1mM ATM PAP 10 min] #id11 [RNaseIII (0.08U) 5min]/[1mM:0.01mM ATM:3'dATP PAP 10 min] #id12 [RNaseIII (0.08U) 5min]/[1mM:0.05mM ATM:3'dATP PAP 10 min] #id13 [RNaseIII (0.08U) 5min]/[1mM:0.1mM ATM:3'dATP PAP 10 min] #id14 [RNaseIII (0.04U) 5min]/[1mM:0.05mM ATM:3'dATP PAP 10 min] #id15 [RNaseIII (0.01U) 5min]/[1mM:0.05mM ATM:3'dATP PAP 10 min] #id16 No RNaseIII/no PAP #id17 NC Results: 3'dATP again increases the proportion of the library falling around 200bp. Does this indicate over-fragmentation? Reducing the amount of RNaseIII from 0.08U to 0.01U did not seem to affect the distribution... may represent the bias for PAP on the smaller fragments over larger ones? If that's the case then 3'dATP may not be helping prevent this bias... I should have added PAP to the unfragmented (no RnaseIII) condition to help confirm that this ~200bp band is due to the fragmentation. == Final Library Preparation == *Beads purified twice at (0.8x), re-suspend in 10ul
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