Blue:RNA-Seq Experiments:01132014: Difference between revisions
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>B1lake (Created page with "== RNA-Seq Library Information == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Samples''' | ali...") |
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| align="center" style="background:#f0f0f0;"|'''Library Conc''' | | align="center" style="background:#f0f0f0;"|'''Library Conc''' | ||
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| BL_totoRNAseq_10pgUHRR_Jan13||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_PE_R||N2. | | BL_totoRNAseq_10pgUHRR_Jan13||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_PE_R||N2.id15||totoRNAseq|||| | ||
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| || | | ||hNPC||SC - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_id1-10||N2.id16||totoRNAseq|||| | ||
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| ||hNPC||SC - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_id1-10||N2.id17||totoRNAseq|||| | |||
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| ||hNPC||10 cells - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_id1||N2.id18||totoRNAseq|||| | |||
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Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence] | Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence] | ||
== Purpose == | == Purpose == |
Revision as of 21:28, 13 January 2014
RNA-Seq Library Information
Library ID | Samples | Input RNA | TSO Primer | Bar Code Primers | Bar Code Primer 2 | Type of Seq | Reads | Library Conc |
BL_totoRNAseq_10pgUHRR_Jan13 | UHRR/ERCC | 10pg - RNase3 15s/Affymetrix PAP 4min | TSO.r06 | T20VN_PE_R | N2.id15 | totoRNAseq | ||
hNPC | SC - RNase3 15s/Affymetrix PAP 4min | TSO.r06 | T20VN_id1-10 | N2.id16 | totoRNAseq | |||
hNPC | SC - RNase3 15s/Affymetrix PAP 4min | TSO.r06 | T20VN_id1-10 | N2.id17 | totoRNAseq | |||
hNPC | 10 cells - RNase3 15s/Affymetrix PAP 4min | TSO.r06 | T20VN_id1 | N2.id18 | totoRNAseq |
Primer sequences [1]
Purpose
- Test optimized RNase III and PAP conditions on 10pg RNA based on 01082014
- Use ERCCs to determine level of transcript amplification between libraries
- Combine libraries prior to TSO and amplification to reduce variability between samples and amplification cycle number
- Compare with single cell sorted samples ([2])
RNAseIII-Fragmented Library Preparation
- RNA
Volume 0.02ng/ul 0.5ul 1:10E6 0.2ul 0.1uM T20VN 0.1ul dH2O 0.2ul Total 1ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1 min
Volume 10x RNaseIII buffer 0.2ul RNase III 0.1ul dH20 0.7ul Total 2ul
- Incubate @ 37C 15s-20s
- 3' Tailing
Volume 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 4 min
- Add 1ul of 0.1uM T20VN
- Reverse Transcription
Volume 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, combine wells
- 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
Volume 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)
Volume TSO Reaction 10ul 2x Buffer 25ul 10uM P1-STRT 1ul 10uM PCR_R_N2_id 1ul H2O 13ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 9x (1ng); 15x (10pg) 72C, 5min
Results of Amplification
- Run 5ul on gel:
Results:
Final Library Preparation
- Beads purified twice at (0.8x), re-suspended in 10ul
- PCR on 0.5ul each library using ILMN_PCR_F and either ERCC130R227 or ERCC130R964.
Results: