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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_N2id_89||huES8||Single cell||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id1-24 (no 17)]||[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|| |- | BL_huES8_SingleCell_N2id_90||UHRR||250pg||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id17]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id90]||totoRNAseq||Read1 plus N2 barcode read and Read2 for T20 barcode read|| |- |} == 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 10pg(9)||UHRR 250pg(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)||10cells(24) |- | |} 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 == *Generate totoRNAseq single cell libraries using modified Smarter TSO (r06) as done in [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08202013 08202013] that involved r04 *Compare r04 with r06 == RNAseIII-Fragmented Library Preparation == :'''Cell Sort''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA 250pg or Single Cells||||||||||- |- |1x Lysis Buffer||||||||||0.4ul |- |Total||||||||||0.4ul |- |} ''Note: add in UHRR control at this point'' :'''Lysis''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA||||||||||0.4ul |- |10x RNaseIII Buffer||||||||||0.2ul |- |0.1uM T20V.id||||||||||0.1ul |- |dH20||||||||||0.55ul |- |Total||||||||||1.25ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1min'' :::''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> |- |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 |- |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)||||||||||0.1ul |- |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 ::#Remove supernatant and wash 2x with 1ml 80% EtOH ::#Air Dry 10min or until cracking first occurs ::#Resuspend beads in 10ul dH2O, incubate 2min, transfer to new tube - vacuspin 8 min to bring to 3ul :'''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'' :'''USER''' ::''Add 1ul and incubate at 37C for 30 min'' :'''PCR (PhusionHF)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||11ul |- |2x Buffer||||||||||25ul |- |10uM P1-STRT||||||||||1ul |- |10uM PCR_R_N2_id89,90||||||||||1ul |- |H2O||||||||||12ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 72C 20s||||||||||11x |- |72C, 5min |- |} == Results of Amplification == *Run 5ul on gel: [[File:10012013_PCR1_gel1.jpg]] == Final Library Preparation == *Beads purify (0.8x), re-suspend in 10ul *Run 0.5ul on gel: [[File:10012013_PCR1_gel2.jpg]] *PCR of 0.5ul using ILMN_PCR_F/R: [[File:10012013_PCR2_PCRF-R.jpg]] *PCR of 0.5ul using ILMN_PCR_F/ERCC130_R227: [[File:10012013_PCR2_ERCC.jpg]] Note: *Pre-PCR TSO control is RnaseIII Smarter TSO control from 09102013 *To add both 10pg and 250pg UHRR to the plate, lysis buffer (containing ERCC) was removed and 0.4ul UHRR was added - the ERCC expression for id90 should be much smaller than id89 (which has 23 combined wells...)
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