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Blue:RNA-Seq Experiments:totoRNAseq RNaseII Test
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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_huES8_SingleCell_||huES8||Single cell||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r04]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id1-96]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id81-82]||totoRNAseq||Read1 plus N2 barcode read and Read2 for T20 barcode read|| |- | ||huES8||5-10 cells|||||||||||| |- | ||UHRR||10pg|||||||||||| |- | |} == Plate Layout: Samples and Indexes == {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' | align="center" style="background:#f0f0f0;"|'''4''' | align="center" style="background:#f0f0f0;"|'''5''' | align="center" style="background:#f0f0f0;"|'''6''' | align="center" style="background:#f0f0f0;"|'''7''' | align="center" style="background:#f0f0f0;"|'''8''' | align="center" style="background:#f0f0f0;"|'''9''' | align="center" style="background:#f0f0f0;"|'''10''' | align="center" style="background:#f0f0f0;"|'''11''' | align="center" style="background:#f0f0f0;"|'''12''' |- | align="center" style="background:#f0f0f0;"|'''A'''||NC(1)||UHRR(9)||Nc(17)||UHRR(25)||Nc(33)||UHRR(41)||Nc(49)||UHRR(57)||Nc(65)||UHRR(73)||Nc(81)||UHRR(89) |- | align="center" style="background:#f0f0f0;"|'''B'''||Sc(2)||Sc(10)||Sc(18)||Sc(26)||Sc(34)||Sc(42)||Sc(50)||Sc(58)||Sc(66)||Sc(74)||Sc(82)||Sc(90) |- | align="center" style="background:#f0f0f0;"|'''C'''||Sc(3)||Sc(11)||Sc(19)||Sc(27)||Sc(35)||Sc(43)||Sc(51)||Sc(59)||Sc(67)||Sc(75)||Sc(83)||Sc(91) |- | align="center" style="background:#f0f0f0;"|'''D'''||Sc(4)||Sc(12)||Sc(20)||Sc(28)||Sc(36)||Sc(44)||Sc(52)||Sc(60)||Sc(68)||Sc(76)||Sc(84)||Sc(92) |- | align="center" style="background:#f0f0f0;"|'''E'''||Sc(5)||Sc(13)||Sc(21)||Sc(29)||Sc(37)||Sc(45)||Sc(53)||Sc(61)||Sc(69)||Sc(77)||Sc(85)||Sc(93) |- | align="center" style="background:#f0f0f0;"|'''F'''||Sc(6)||Sc(14)||Sc(22)||Sc(30)||Sc(38)||Sc(46)||Sc(54)||Sc(62)||Sc(70)||Sc(78)||Sc(86)||Sc(94) |- | align="center" style="background:#f0f0f0;"|'''G'''||Sc(7)||Sc(15)||Sc(23)||Sc(31)||Sc(39)||Sc(47)||Sc(55)||Sc(63)||Sc(71)||Sc(79)||Sc(87)||Sc(95) |- | align="center" style="background:#f0f0f0;"|'''H'''||Sc(8)||10cells(16)||Sc(24)||10cells(32)||Sc(40)||10cell(48)||Sc(56)||10 cell(64)||Sc(72)||10cells(80)||Sc(88)||10cells(96) |- | |} Note: SC=Single cell; UHRR=Universal Human Reference RNA Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08122013 08122013] == Purpose == *Repeat Single cell totoRNAseq protocol last used on 06142013b **Test Decrease in T20 concentration from 0.2uM to 0.1uM **Test Decrease in total reaction volume == Library Preparation (totoRNAseq): == :'''Cell Sort''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA 10pg or Single Cells||||||||||- |- |1x Lysis Buffer||||||||||0.5ul |- |Total||||||||||0.5ul |- |} :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA||||||||||0.5ul |- |10x RNaseIII Buffer||||||||||0.15ul |- |0.1uM T20V.id||||||||||0.1ul |- |dH20||||||||||0.5ul |- |Total||||||||||1.25ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1min (Centrifuge 1 min during this incubation and add '''UHRR''')'' :::''Incubate @ 4C 10min'' :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA||||||||||1.25ul |- |RNase III||||||||||0.08ul |- |dH20||||||||||0.67ul |- |Total||||||||||2ul |- |} :::''Incubate @ 37C 10 min'' :::''Add 1.1ul dH2O :::''Incubate @ 65C 10 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PNK_fRNA||||||||||3.1ul |- |5x RT Buffer||||||||||0.5ul |- |1mM ATP||||||||||0.25ul |- |0.5U/ul PAP (1:10d in H2O)||||||||||0.25ul |- |} :::''Incubate @ 37C 5 min'' :::''Transfer to Ice'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA||||||||||4.1ul |- |10mM dNTP Mix||||||||||1ul |- |dH2O||||||||||0ul |- |0.1uM T20V.ID||||||||||0.9ul |- |} :::''Incubate @ 65C 3 min'' :::''Transfer to Ice'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5xRT Buffer||||||||||1.5ul |- |100mM DTT||||||||||0.17ul |- |Betaine (5M)||||||||||2ul |- |RNase Inhibitor (40U/ul)||||||||||0.33ul |- |Smarter MMLV (200U/ul)||||||||||0.5ul |- |Total||||||||||10ul |- |} :::''Incubate @ 42C 30 min'' :'''Beads Purification''' ::#Add 15ul beads per well ::#Allow to bind 15 min ::#Combine all odd or even column wells ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry 10min or until cracking first occurs ::#Resuspend beads in 20ul dH2O, incubate 2min, transfer to new tube ::#Vacuspin room temperature for 10+min to bring volume to 6ul :'''TSO (x2)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Purified cDNA||||||||||3ul |- |5x RT buffer [MMLV]||||||||||2ul |- |10mM dNTP mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |10uM TSO.r04||||||||||1ul |- |Betaine (5M)||||||||||2ul |- |RNase Inhibitor||||||||||0.25ul |- |Smarter MMLV||||||||||0.5ul |- |Total||||||||||10ul |- |} :::''Incubate @42C 10min'' :::''Incubate @70C 10min'' :'''USER''' ::''Add 1ul and incubate at 37C for 30 min'' :'''PCR (PhusionHF)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||11ul |- |2x Buffer||||||||||25ul |- |10uM PB_PCR_Fmod||||||||||1ul |- |10uM PCR_N2_idX||||||||||1ul |- |H2O||||||||||12ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 72C 20s||||||||||15x |- |72C, 5min |- |} :::*Ampure Beads Purify (0.8x) :::*Resuspend in 50ul :::*Ampure Beads Purify (0.8x) :::*Resuspend in 10ul :::*Run 1ul on gel == Results of Amplification == *Ran 1ul on gel: [[File:08072013_PCR1_gel1.jpg]] N2.id81 = Odd Wells N2.id82 = Even Wells == Final Library Preparation == *Re-Amplified 1ul using Sybr-Green and ILMN_PCR_F/R ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Library||||||||||1ul |- |2xSybr Buffer||||||||||25ul |- |10uM ILMN_PCR_F[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence]||||||||||1ul |- |10uM ILMN_PCR_R[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence]||||||||||1ul |- |dH2O||||||||||22ul |- |Total||||||||||50ul |- |} :::''95C 30sec'' :::''95C 10s -> 60C 20s -> 72C 30s'' => 5X :::''72C, 5min'' *Ampure Beads purified (0.8x) *Resuspended in 10ul and ran 1 ul on gel: [[File:08072013_PCR2_gel1.jpg]] Re-amplified 0.5ul library using un-modified ILMN_PCR_F/R and ran 5ul of 50 on gel:
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