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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_totoRNAseq_1ngUHRR_Dec03||UHRR/ERCC||1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id81-82||totoRNAseq|||| |- | ||UHRR/ERCC||100pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id83-84||totoRNAseq|||| |- | ||UHRR/ERCC||10pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id385-86||totoRNAseq|||| |- | ||UHRR/ERCC||1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r07||T20VN_PE_R||N2.id87-88||totoRNAseq|||| |- | ||UHRR/ERCC||1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step||TSO.r06||T20VN_PE_R||N2.id89-90||totoRNAseq|||| |- | ||UHRR/ERCC||100pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step||TSO.r06||T20VN_PE_R||N2.id91-92||totoRNAseq|||| |- | ||UHRR/ERCC||10pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step||TSO.r06||T20VN_PE_R||N2.id93-94||totoRNAseq|||| |- | ||UHRR/ERCC||1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step||TSO.r07||T20VN_PE_R||N2.id95-96||totoRNAseq|||| |- |} Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence] == Purpose == *Test efficiency of Affymetrix PAP to generate libraries from decreasing amounts of RNA from 1ng down to 10pg *Use ERCCs to determine level of transcript amplification between libraries - ERCC amplification using previous version of the totoRNAseq protocol from 09-27-13 showed progressively lower ERCC quantities in the final libraries from 100pg and 10pg UHRR == RNAseIII-Fragmented Library Preparation == :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |UHRR 2ng/ul; 0.2ng/ul; 0.02ng/ul ||||||||||0.5ul |- |ERCC 1:10E4; 1:10E5; 1:10E6||||||||||0.2ul |- |0.1uM T20VN||||||||||0.1ul |- |dH2O||||||||||0.2ul |- |Total||||||||||1ul |- |} :::''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 5 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 10 min'' :::''Add 1ul of 0.1uM T20VN [for 1-step: 0.1ul 1mM T20VN + 0.9ul 10uM TSO]'' :'''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 30 min'' :'''Beads Purification (2-step only)''' ::#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 Reaction (2-step only)''' ::{| {{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||||||||||9x (1ng) 12x (100pg) 15x (10pg) |- |72C, 5min |- |} == Results of Amplification == *Run 5ul on gel: [[File:12032013_PCR1_gel1.jpg]] Results: Can see that the Affymetrix PAP protocol loses efficiency with decreasing RNA - may not be sensitive enough. R07 on 1ng looks very poor - too inefficient. 1-step does not appear to be an option, r07 in this context does not seem to be affected by PAP, but clearly works poorly for template switching. == Final Library Preparation == *Beads purified twice at (0.8x), re-suspend in 10ul See [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:12062013#Final_Library_Preparation 12062013] for gel image of final library (1ng)
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