Blue:RNA-Seq Experiments:totoRNAseq optimizations: Difference between revisions
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== Experimental Overview == | == Experimental Overview == | ||
[[File:TotoRNAseq expanded optimization.jpg]] | [[File:TotoRNAseq expanded optimization.jpg|600px]] | ||
== Purpose == | == Purpose == |
Revision as of 01:36, 11 February 2014
Experimental Overview
File:TotoRNAseq expanded optimization.jpg
Purpose
- Systematic analysis of different methods for addition of forward ILMN sequencing adapter to totoRNAseq libraries
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_1ngUHRR_ | UHRR/ERCC | 4x1ng - Mg Frag/Enzymatics PAP 5 min - STRT-Seq | TSO.r06 | T20VN_r01 | N2.id1 | totoRNAseq | ||
UHRR/ERCC | 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq | TSO.r06 | T20VN_r01 | N2.id2 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq | xxx | T20VN_r01 | N2.id3 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Affymetrix PAP 10 min - Quartz-Seq | xxx | T20VN_r01 | N2.id4-7 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Enzymatics PAP 5 min - Cel-Seq | xxx | T20VN_r01 | N2.id3 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Affymetrix PAP 10 min - Cel-Seq | xxx | T20VN_r01 | N2.id4-7 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Enzymatics PAP 5 min - Nugen | xxx | T20VN_r01 | N2.id3 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Affymetrix PAP 10 min - Nugen | xxx | T20VN_r01 | N2.id4-7 | totoRNAseq |
Primer sequences [1]
RNAseIII-Fragmented Library Preparation
- RNA
Volume 2ng/ul 0.5ul 1:104 ERCC 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 1 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.id
- 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 ~8min to 3ul
STRT-Seq Library Preparation
- 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) 72C, 5min
- Run 5ul on gel:
Results: