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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||10ng - RNase3 (5mM MnCl2)/PAP (2.5mM MnCl2) 10min ATP||TSO.r06||T20VN_PE_R||N2.id81||totoRNAseq|||| |- | BL_||UHRR/ERCC||10ng - RNase3 (5mM MnCl2)/PAP (2.5mM MnCl2) 10min ATP/dATP||TSO.r06||T20VN_PE_R||N2.id82||totoRNAseq|||| |- | BL_||UHRR/ERCC||10ng - RNase3 (5mM MnCl2)/PAP (2.5mM MnCl2) 30min ATP/dATP||TSO.r06||T20VN_PE_R||N2.id83||totoRNAseq|||| |- | BL_||UHRR/ERCC||10ng - RNase3 (10mM MnCl2)/PAP (5mM MnCl2) 10min ATP||TSO.r06||T20VN_PE_R||N2.id84||totoRNAseq|||| |- | BL_||UHRR/ERCC||10ng - RNase3 (10mM MnCl2)/PAP (5mM MnCl2) 10min ATP/dATP||TSO.r06||T20VN_PE_R||N2.id85||totoRNAseq|||| |- | BL_||UHRR/ERCC||10ng - RNase3 (10mM MnCl2)/PAP (5mM MnCl2) 30min ATP/dATP||TSO.r06||T20VN_PE_R||N2.id86||totoRNAseq|||| |- | BL_||UHRR/ERCC||10ng - RNase3 (20mM MnCl2)/PAP (10mM MnCl2) 10min ATP||TSO.r06||T20VN_PE_R||N2.id87||totoRNAseq|||| |- | BL_||UHRR/ERCC||10ng - RNase3 (20mM MnCl2)/PAP (10mM MnCl2) 10min ATP/dATP||TSO.r06||T20VN_PE_R||N2.id88||totoRNAseq|||| |- | BL_||UHRR/ERCC||10ng - RNase3 (20mM MnCl2)/PAP (10mM MnCl2) 30min ATP/dATP||TSO.r06||T20VN_PE_R||N2.id89||totoRNAseq|||| |- |} == Purpose == *Full totoRNAseq protocol on samples prepared similarly to [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:11062013 11-06-2013] *Determine optimal PAP conditions for library production *Use T20VN to limit the primer annealing to the ends flanking RNA fragments rather than randomly along extensive poly(A) tails == RNAseIII-Fragmented Library Preparation == :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |UHRR 20ng/ul ||||||||||0.5ul |- |ERCC 1:10E3||||||||||0.2ul |- |1uM T20VN||||||||||0.1ul |- |dH2O||||||||||0.2ul |- |Total||||||||||1ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1 min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5x Ambion PAP buffer||||||||||0.08ul |- |MnCl2||||||||||0.4ul |- |RNase III||||||||||0.1ul |- |dH20||||||||||0.42ul |- |Total||||||||||2ul |- |} :::''Incubate @ 37C 5 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||2ul |- |5x Ambion PAP Buffer||||||||||0.72ul |- |1mM ATP (or mix)||||||||||0.25ul |- |2U/ul PAP||||||||||0.2ul |- |40U/ul Rnase Inhibitor||||||||||0.2ul |- |dH2O||||||||||0.63ul |- |Total||||||||||4ul |- |} :::''Incubate @ 37C 10 min - 30 min'' :::''Add 0.5ul of 2uM T20VN'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||4.5ul |- |5xRT Buffer||||||||||2ul |- |10mM dNTP Mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |Betaine (5M)||||||||||2ul |- |Smarter MMLV (200U/ul)||||||||||0.25ul |- |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 3ul dH2O, incubate 2min, transfer to new tube :'''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||||||||||8x |- |72C, 5min |- |} == Results of Amplification == *Run 5ul on gel: [[File:11122013_PCR1_gel1.jpg]] *Re-Amplified 1ul using ILMN_PCR_F/R and SYBR *Ran 5ul on gel: [[File:11122013_PCR2_gel1.jpg]] Results: Looks like there is ultimately a problem with the altered protocol. It's possible that while MnCl2 allows for more PAP control, it inhibits subsequent RT reactions. The original PCR smear appears too high and upon re-amplification it drops quite low... This similarly high smear was also observed on 11012013 - at that time I miss-interpreted the results as indicating a lack of fragmentation, but instead I now think that the MnCl2 might be blocking subsequent steps. Its possible that rather than focusing on controlling PAP, we should instead focus on preventing accidental fragmentation of poly(A) tailed RNA (i.e. no heating prior to RT reaction). == Final Library Preparation == *Beads purified twice at (0.8x), re-suspend in 10ul
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