Blue:RNA-Seq Experiments:totoRNAseq optimizations: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''Library Conc''' | | align="center" style="background:#f0f0f0;"|'''Library Conc''' | ||
|- | |- | ||
| BL_totoRNAseq_1ngUHRR_||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - STRT-Seq||TSO.r06|| | | BL_totoRNAseq_1ngUHRR_||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - STRT-Seq||TSO.r06||T20VN_id1-4||N2.id1||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq||TSO.r06|| | | ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq||TSO.r06||T20VN_id5-8||N2.id2||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq||TSO.r06|| | | ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq||TSO.r06||T20VN_id9-18||N2.id3||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq||TSO.r06|| | | ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq||TSO.r06||T20VN_id1-4||N2.id4||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq||TSO.r06|| | | ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq||TSO.r06||T20VN_id5-8||N2.id5||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq||TSO.r06|| | | ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq||TSO.r06||T20VN_id9-18||N2.id6||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq||xxx|| | | ||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq||xxx||T20VN_id1-4||N2.id7||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq||xxx|| | | ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq||xxx||T20VN_id5-8||N2.id8||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq||xxx|| | | ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq||xxx||T20VN_id9-18||N2.id9||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq||xxx|| | | ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq||xxx||T20VN_id1-4||N2.id10||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq||xxx|| | | ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq||xxx||T20VN_id5-8||N2.id11||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq||xxx|| | | ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq||xxx||T20VN_id9-18||N2.id12||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - Cel-Seq||xxx|| | | ||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - Cel-Seq||xxx||T20VN_id1-4||N2.id13||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq||xxx|| | | ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq||xxx||T20VN_id5-8||N2.id14||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq||xxx|| | | ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq||xxx||T20VN_id9-18||N2.id15||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq||xxx|| | | ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq||xxx||T20VN_id1-4||N2.id16||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq||xxx|| | | ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq||xxx||T20VN_id5-8||N2.id17||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq||xxx|| | | ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq||xxx||T20VN_id9-18||N2.id18||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - Nugen||xxx|| | | ||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - Nugen||xxx||T20VN_id1-4||N2.id19||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - Nugen||xxx|| | | ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - Nugen||xxx||T20VN_id5-8||N2.id20||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - Nugen||xxx|| | | ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - Nugen||xxx||T20VN_id9-18||N2.id21||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Nugen||xxx|| | | ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Nugen||xxx||T20VN_id1-4||N2.id22||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen||xxx|| | | ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen||xxx||T20VN_id5-8||N2.id23||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen||xxx|| | | ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen||xxx||T20VN_id9-18||N2.id24||totoRNAseq|||| | ||
|- | |- | ||
|} | |} |
Revision as of 02:06, 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
- Comparison of Mg-based and RNaseIII based fragmentation methods
- Comparison of Enzymatics and Affymetrix PAP
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_id1-4 | N2.id1 | totoRNAseq | ||
UHRR/ERCC | 4x100pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq | TSO.r06 | T20VN_id5-8 | N2.id2 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq | TSO.r06 | T20VN_id9-18 | N2.id3 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq | TSO.r06 | T20VN_id1-4 | N2.id4 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq | TSO.r06 | T20VN_id5-8 | N2.id5 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq | TSO.r06 | T20VN_id9-18 | N2.id6 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq | xxx | T20VN_id1-4 | N2.id7 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq | xxx | T20VN_id5-8 | N2.id8 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq | xxx | T20VN_id9-18 | N2.id9 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq | xxx | T20VN_id1-4 | N2.id10 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq | xxx | T20VN_id5-8 | N2.id11 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq | xxx | T20VN_id9-18 | N2.id12 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Enzymatics PAP 5 min - Cel-Seq | xxx | T20VN_id1-4 | N2.id13 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq | xxx | T20VN_id5-8 | N2.id14 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq | xxx | T20VN_id9-18 | N2.id15 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq | xxx | T20VN_id1-4 | N2.id16 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq | xxx | T20VN_id5-8 | N2.id17 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq | xxx | T20VN_id9-18 | N2.id18 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - Mg Frag/Enzymatics PAP 5 min - Nugen | xxx | T20VN_id1-4 | N2.id19 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - Mg Frag/Enzymatics PAP 5 min - Nugen | xxx | T20VN_id5-8 | N2.id20 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - Mg Frag/Enzymatics PAP 5 min - Nugen | xxx | T20VN_id9-18 | N2.id21 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Nugen | xxx | T20VN_id1-4 | N2.id22 | totoRNAseq | |||
UHRR/ERCC | 4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen | xxx | T20VN_id5-8 | N2.id23 | totoRNAseq | |||
UHRR/ERCC | 10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen | xxx | T20VN_id9-18 | N2.id24 | 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: