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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_LCM_10Cell_N2id1_Oct03||Mouse Pyramidal|| 10 cell(C1) - water 70C 5min||[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.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id1]||totoRNAseq||Read1 plus N2 barcode read || |- | BL_LCM_10Cell_N2id2_Oct03||Mouse Pyramidal|| 10 cell(C3) - water 70C 5min||[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.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id2]||totoRNAseq||Read1 plus N2 barcode read || |- | BL_LCM_10Cell_N2id3_Oct03||Mouse Pyramidal|| 10 cell(T1) - Tritonx100 55C 30min||[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.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id3]||totoRNAseq||Read1 plus N2 barcode read || |- | BL_LCM_10Cell_N2id4_Oct03||Mouse Pyramidal|| 10 cell(T4) - Tritonx100 55C 30min||[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.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id4]||totoRNAseq||Read1 plus N2 barcode read || |- | BL_LCM_10Cell_N2id5_||Mouse Pyramidal|| 10 cell(1) - Extraction||[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.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id5]||totoRNAseq||Read1 plus N2 barcode read || |- | BL_LCM_5Cell_N2id6_||Mouse Pyramidal|| 5 cell(1) - Extraction||[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.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id6]||totoRNAseq||Read1 plus N2 barcode read || |- | BL_LCM_100pgHC_N2id7_Oct03||Mouse Pyramidal|| Handcut(2) - Extraction||[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.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id7]||totoRNAseq||Read1 plus N2 barcode read || |- | BL_LCM_100pgHC_N2id8_Oct03||Mouse Pyramidal|| Handcut(2) - Extraction||[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.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id8]||totoRNAseq||Read1 plus N2 barcode read || |- |} LCM samples collected 10022013 (handcut 2 1.4ng/ul) == Purpose == *Generate totoRNAseq libraries using RNaseIII digestion on LCM collected samples of mouse brain pyramidal neurons *Use LCM samples that have been extracted for RNA as control == RNAseIII-Fragmented Library Preparation == :'''Sample Prep''' *Dilute handcut to 133pg/ul (1:10 dilution) *Samples collected in 10ul water or Triton-X100 *Vacuspin to bring volume to 1.0ul Note: :*15 min at RT :**plus 5min at 30C for Extracted (5 cell actually dried out - added 1ul water to try and re-hydrate) :**plus 7min for C1,C3,T1,T4 ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA||||||||||1.0ul |- |ERCC 1:10E5||||||||||0.2ul |- |10x RNaseIII Buffer||||||||||0.2ul |- |0.1uM T20V.id2||||||||||0.1ul |- |Total||||||||||1.5ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1min'' :::''Incubate @ 4C 1min'' :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA||||||||||1.5ul |- |RNase III||||||||||0.08ul |- |dH20||||||||||0.42ul |- |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.ID2 ||||||||||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 ::#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 5 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'' :'''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||||||||||12x |- |72C, 5min |- |} == Results of Amplification == *Run 5ul on gel: [[File:10032013_PCR1_gel1.jpg]] == Final Library Preparation == *Beads purify (0.8x), re-suspend in 50ul *Beads purify again at 0.8x (if primer dimer band is apparent) *Resuspend beads in 10ul *Run 1ul on gel: [[File:10032013_PCR1_gel2.jpg]]
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