Blue:RNA-Seq Experiments:11132013
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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 | 10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP | TSO.r06 | T20VN_PE_R | N2.id90 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/3'dATP (1:0.1) | TSO.r06 | T20VN_PE_R | N2.id91 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/dATP (1:1) | TSO.r06 | T20VN_PE_R | N2.id92 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP | TSO.r07 | T20VN_PE_R | N2.id93 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/3'dATP (1:0.1) | TSO.r06 | T20VN_PE_R | N2.id90 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/dATP (1:1) | TSO.r06 | T20VN_PE_R | N2.id91 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP then 5min 3'dATP | TSO.r06 | T20VN_PE_R | N2.id92 | totoRNAseq | ||
BL_ | UHRR/ERCC | 10ng - RNase3 (Affy buffer)/Affymetrix PAP 10min ATP then 5min 3'dATP | TSO.r06 | T20VN_PE_R | N2.id93 | totoRNAseq |
Purpose
- Full totoRNAseq protocol on samples prepared similarly to 11-06-2013
- Determine optimal PAP conditions for library production
- Use T20VN to limit the primer annealing to the ends flanking RNA fragments rather than randomly along extensive poly(A) tails
RNAseIII-Fragmented Library Preparation
- RNA
Volume UHRR 20ng/ul 0.5ul ERCC 1:10E3 0.2ul 1uM T20VN 0.1ul dH2O 0.2ul Total 1ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1 min
Volume 5x Ambion PAP buffer 0.08ul MnCl2 0.4ul RNase III 0.1ul dH20 0.42ul Total 2ul
- Incubate @ 37C 5 min
- 3' Tailing
Volume fRNA 2ul 5x Ambion PAP Buffer 0.72ul 1mM ATP (or mix) 0.25ul 2U/ul PAP 0.2ul 40U/ul Rnase Inhibitor 0.2ul dH2O 0.63ul Total 4ul
- Incubate @ 37C 10 min - 30 min
- Add 0.5ul of 2uM T20VN
- Reverse Transcription
Volume PAP RNA 4.5ul 5xRT Buffer 2ul 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 3ul dH2O, incubate 2min, transfer to new tube
- TSO
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 8x 72C, 5min
Results of Amplification
- Run 5ul on gel:
- Re-Amplified 1ul using ILMN_PCR_F/R and SYBR
- Ran 5ul on gel:
Final Library Preparation
- Beads purified twice at (0.8x), re-suspend in 10ul