Blue:RNA-Seq Experiments:01292014: Difference between revisions
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| BL_totoRNAseq_10pgUHRR_Jan29||UHRR/ERCC||10x10pg - 0.05U RNase3 1min/PAP 4min/RT 42C 30min||TSO.r06||T20VN_id1-10||N2.id1||totoRNAseq|||| | | BL_totoRNAseq_10pgUHRR_Jan29||UHRR/ERCC||10x10pg - 0.05U RNase3 1min/PAP 4min/RT 42C 30min||TSO.r06||T20VN_id1-10||N2.id1||totoRNAseq|||| | ||
|- | |- | ||
| ||hNPC/ERCC||10xSC - 0.05U RNase3 1min/PAP 4min/RT | | ||hNPC/ERCC||10xSC - 0.05U RNase3 1min/PAP 4min/RT 37C 60min||TSO.r06||T20VN_id1-10||N2.id2||totoRNAseq|||| | ||
|- | |- | ||
| ||hNPC/ERCC||10xSC - 0.05U RNase3 1min/PAP 4min/RT 37C 60min||TSO.r06||T20VN_id1-10||N2.id3||totoRNAseq|||| | | ||hNPC/ERCC||10xSC - 0.05U RNase3 1min/PAP 4min/RT 37C 60min||TSO.r06||T20VN_id1-10||N2.id3||totoRNAseq|||| | ||
|- | |- | ||
| ||UHRR/ERCC||4x1ng - 0.1U RNase3 1min/PAP 10min/RT 42C 30min||TSO.r06|| | | ||UHRR/ERCC||4x1ng - 0.1U RNase3 1min/PAP 10min/RT 42C 30min||TSO.r06||T20VN_r01||N2.id4-8||totoRNAseq|||| | ||
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Revision as of 00:28, 30 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_Jan29 | UHRR/ERCC | 10x10pg - 0.05U RNase3 1min/PAP 4min/RT 42C 30min | TSO.r06 | T20VN_id1-10 | N2.id1 | totoRNAseq | ||
hNPC/ERCC | 10xSC - 0.05U RNase3 1min/PAP 4min/RT 37C 60min | TSO.r06 | T20VN_id1-10 | N2.id2 | totoRNAseq | |||
hNPC/ERCC | 10xSC - 0.05U RNase3 1min/PAP 4min/RT 37C 60min | TSO.r06 | T20VN_id1-10 | N2.id3 | totoRNAseq | |||
UHRR/ERCC | 4x1ng - 0.1U RNase3 1min/PAP 10min/RT 42C 30min | TSO.r06 | T20VN_r01 | N2.id4-8 | totoRNAseq |
Note: hNPCs from [1] - excluding the 10pg control, 0 cell controls and 10 cell control. Combined 10 cells per N2 index for ~100pg total RNA
Primer sequences [2]
Purpose
- Test optimized RNase III and PAP conditions on 10pg RNA based on 01222014
- 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 ([3])
RNAseIII-Fragmented Library Preparation
- RNA
Volume 2ng/ul or 0.02ng/ul or NPC 0.5ul 1:104 or 1:10E6 ERCC (UHRR only) 0.2ul 0.1uM T20VN 0.1ul dH2O 0.2ul Total 1ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1 min
- Incubate @ 4C 10 min
Volume 10x RNaseIII buffer 0.2ul RNase III 0.1ul or 0.05ul dH20 0.7ul or 0.75ul 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 4 min or 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 or 37C for 60 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
- 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) or 12x (100pg) 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: