Blue:RNA-Seq Experiments:12032013: Difference between revisions
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:::''Add 1ul of 0.1uM T20VN'' | :::''Add 1ul of 0.1uM T20VN [for 1-step: 0.1ul 1mM T20VN + 0.9ul 10uM TSO]'' | ||
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== Results of Amplification == | == Results of Amplification == |
Revision as of 23:17, 4 December 2013
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_ | UHRR/ERCC | 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP | TSO.r06 | T20VN_PE_R | N2.id81-82 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP | TSO.r06 | T20VN_PE_R | N2.id83-84 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP | TSO.r06 | T20VN_PE_R | N2.id385-86 | totoRNAseq | ||
BL_ | UHRR/ERCC | 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP | TSO.r07 | T20VN_PE_R | N2.id87-88 | totoRNAseq | ||
BL_ | UHRR/ERCC | 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step | TSO.r06 | T20VN_PE_R | N2.id89-90 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step | TSO.r06 | T20VN_PE_R | N2.id91-92 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step | TSO.r06 | T20VN_PE_R | N2.id93-94 | totoRNAseq | ||
BL_ | UHRR/ERCC | 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step | TSO.r07 | T20VN_PE_R | N2.id95-96 | totoRNAseq |
Purpose
- Test efficiency of Affymetrix PAP to generate libraries from decreasing amounts of RNA from 1ng down to 10pg
- Use ERCCs to determine level of transcript amplification between libraries - ERCC amplification using previous version of the totoRNAseq protocol from 09-27-13 showed progressively lower ERCC quantities in the final libraries from 100pg and 10pg UHRR
RNAseIII-Fragmented Library Preparation
- RNA
Volume UHRR 2ng/ul; 0.2ng/ul; 0.02ng/ul 0.5ul ERCC 1:10E4; 1:10E5; 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 5 min
- 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 10 min
- Add 1ul of 0.1uM T20VN [for 1-step: 0.1ul 1mM T20VN + 0.9ul 10uM TSO]
- 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
- 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) 12x (100pg) 15x (10pg) 72C, 5min
Results of Amplification
- Run 5ul on gel:
Final Library Preparation
- Beads purified twice at (0.8x), re-suspend in 10ul