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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_HSC_AIT2GFP+_N2id89_0828||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.id1-24]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id89]||totoRNAseq||Read1 plus N2 barcode read and Read2 for T20 barcode read|| |- | ||UHRR||250pg||TSO.r04||T20V.id1||N2.id90|||||| |- | ||UHRR||250pg||TSO.r04||T20V.id2||N2.id91|||||| |- |} == 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;"|'''A'''||10cell(1)||10cell(9)||Sc(17) |- | align="center" style="background:#f0f0f0;"|'''B'''||Sc(2)||Sc(10)||Sc(18) |- | align="center" style="background:#f0f0f0;"|'''C'''||Sc(3)||Sc(11)||Sc(19) |- | align="center" style="background:#f0f0f0;"|'''D'''||Sc(4)||Sc(12)||Sc(20) |- | align="center" style="background:#f0f0f0;"|'''E'''||Sc(5)||Sc(13)||Sc(21) |- | align="center" style="background:#f0f0f0;"|'''F'''||Sc(6)||Sc(14)||Sc(22) |- | align="center" style="background:#f0f0f0;"|'''G'''||Sc(7)||Sc(15)||Sc(23) |- | align="center" style="background:#f0f0f0;"|'''H'''||Sc(8)||Sc(16)||Sc(24) |- | |} Note: SC=Single cell => N5_HSC_AIT2 CD34+GFP+; UHRR=Universal Human Reference RNA Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08052013 08052013] == Purpose == *Try Single cell RNAseIII/TSO totoRNAseq protocol on Zhili's HSC sorted cells **Cells are in hiMg based buffer, however RNaseIII protocol looks more promising (08202013) **Test RNaseIII adaptation using hiMg RT sort buffer == Library Preparation (totoRNAseq): == :'''Cell Sort''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA 250pg or Single Cells||||||||||- |- |2.5x hiMgRT/Lysis Buffer||||||||||2ul |- |Total||||||||||2ul |- |} ''Note: add in UHRR control at this point'' :'''Cell Lysis''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA||||||||||2ul |- |0.1uM T20V.id||||||||||0.1ul |- |dH20||||||||||0.45ul |- |Total||||||||||2.55ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1min'' :::''Incubate @ 4C 10min'' :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA||||||||||2.55ul |- |RNase III||||||||||0.08ul |- |dH20||||||||||0.47ul |- |Total||||||||||3.1ul |- |} :::''Incubate @ 37C 10 min'' :::''Add 1ul dH2O :::''Incubate @ 65C 10 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PNK_fRNA||||||||||4.1ul |- |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||||||||||4.6ul |- |10mM dNTP Mix||||||||||1ul |- |0.1uM T20V.ID||||||||||0.9ul |- |} :::''Incubate @ 65C 3 min'' :::''Transfer to Ice'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5xRT Buffer||||||||||1ul |- |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 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) or 7.7ul (IVT) == TSO == :'''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 [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence PB_PCR_F]||||||||||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: see below == Final Library Preparation == *Re-Amplified 1ul using Sybr-Green and ILMN_PCR_F/R ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Library||||||||||1ul |- |2xSybr Buffer||||||||||25ul |- |10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence ILMN_PCR_F]||||||||||1ul |- |10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence ILMN_PCR_R]||||||||||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:08282013_PCR1-2_gel1.jpg]]
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