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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_1ng_100pgUHRR_Dec06||UHRR/ERCC||1ng - RNase3 1min (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id9-10||totoRNAseq|||| |- | ||UHRR/ERCC||100pg - RNase3 1min (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id11-12||totoRNAseq|||| |- | ||UHRR/ERCC||10pg - RNase3 1min (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id13-14||totoRNAseq|||| |- | ||UHRR/ERCC||1ng - RNase3 1min (RIII buffer)/Affymetrix PAP 30min ATP||TSO.r06||T20VN_PE_R||N2.id17-18||totoRNAseq|||| |- | ||UHRR/ERCC||100pg - RNase3 1min (RIII buffer)/Affymetrix PAP 30min ATP||TSO.r06||T20VN_PE_R||N2.id19-20||totoRNAseq|||| |- | ||UHRR/ERCC||10pg - RNase3 1min (RIII buffer)/Affymetrix PAP 30min ATP||TSO.r06||T20VN_PE_R||N2.id21-22||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 as done in 12032013, but with less fragmentation by RNaseIII (1 min vs. 5 min) and longer PAP treatment *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 1 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 10-30 min'' :::''Add 1ul of 0.1uM T20VN'' :'''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''' ::#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''' ::{| {{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:12062013_PCR1_gel1.jpg]] Results: Can see better amplification of 100pg and 10pg after shortening the RNAse III digestion time (compared to [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:12032013 12032013]). Increasing PAP time may not be necessary. == Final Library Preparation == *Beads purified twice at (0.8x), re-suspended in 10ul *Test ERCC amplification: **Diluted PCR product 1:5 and used 1ul: ***PCR Product: 1ul ***2x Sybr: 12.5ul ***ILMN_PCR_F: 0.5ul ***ERCC130R227 or R964: 0.5ul ***dH2O: 10.5ul **PCR Results: [[File:12062013_PCR2_ERCC-3.jpg]] [[File:12062013_PCR2_ERCC-5.jpg]] Results: Can see that the reactions for 1ng and 100pg show similar efficiency for amplifying the 3' end of the transcript and only a slight drop in efficiency for the 5' end. 10 pg shows a greater drop in efficiency and you can see more of a bias towards the 3' end. Also, comparing the level of amplification of the transcript in the library with the previous version ([http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:09272013#Final_Library_Preparation 09272013])of the protocol (used 0.5ul final library), this protocol shows a similar level of amplification (used 0.2ul of the final library) for the 5' end of the transcript. It appears that the global level of amplification is reduced in the newer version of the protocol, hopefully this will represent fewer poly-A reads and more specific representation of transcripts. Better recovery of the 10pg RNA might be possible through reduction of RNAse III fragmentation further. Note that the curves above include 10 and 30 min pap treatments - no significant improvement in amplification was observed with the increased PAP time. *Combined: **1203 1ng id81, 82 **1206 1ng id9,10, 17, 18 **1206 100pg id11, 12, 19, 20 **1206 10pg id13, 14, 21, 22 *Ran 1ul on gel: [[File:12062013_PCR1_gel2.jpg]]
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