Blue:RNA-Seq Experiments:01082014
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RNA-Seq Library Information[edit]
Library ID | Samples | Input RNA | TSO Primer | Bar Code Primers | Bar Code Primer 2 | Type of Seq | Reads | Library Conc |
BL_totoRNAseq_10pgUHRR_Jan08 | UHRR/ERCC | 10pg - RNase3 15s/Affymetrix PAP 1min | TSO.r06 | T20VN_PE_R | N2.id1-2 | totoRNAseq | ||
UHRR/ERCC | 10pg - RNase3 15s/Affymetrix PAP 2min | TSO.r06 | T20VN_PE_R | N2.id3-4 | totoRNAseq | |||
UHRR/ERCC | 10pg - RNase3 15s/Affymetrix PAP 4min | TSO.r06 | T20VN_PE_R | N2.id5-6 | totoRNAseq | |||
UHRR/ERCC | 10pg - RNase3 15s/Affymetrix PAP 6min | TSO.r06 | T20VN_PE_R | N2.id7-8 | totoRNAseq | |||
UHRR/ERCC | 10pg - RNase3 15s/Affymetrix PAP 8min | TSO.r06 | T20VN_PE_R | N2.id9-10 | totoRNAseq | |||
UHRR/ERCC | 10pg - RNase3 15s/Affymetrix PAP 10min | TSO.r06 | T20VN_PE_R | N2.id11-12 | totoRNAseq | |||
UHRR/ERCC | 1ng - RNase3 1min/Affymetrix PAP 10min | TSO.r06 | T20VN_PE_R | N2.id13-14 | totoRNAseq |
Primer sequences [1]
Purpose[edit]
- Test optimal PAP treatment time for 10pg RNA based on 12062013
- Use ERCCs to determine level of transcript amplification between libraries
RNAseIII-Fragmented Library Preparation[edit]
- RNA
Volume UHRR 2ng/ul or 0.02ng/ul 0.5ul ERCC 1:10E4 or 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 15s-1min
- 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 1-10 min
- Add 1ul of 0.1uM T20VN
- 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); 15x (10pg) 72C, 5min
Results of Amplification[edit]
- Run 5ul on gel:
Results: Global amplification levels seem a little low, but otherwise looks fine. Some variability with the 10pg that could be due to the beads purification stage following RT...
Final Library Preparation[edit]
- 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.
File:01082014 ERCC130 R227.jpgFile:01082014 ERCC130 R964b.jpg
Results:
- Still variable amplification levels seen with 10pg. More consistency might be achieved by combining 10pg samples for beads purification prior to TSO/PCR
- Can see at least one of the 2min or 4 min showing similar levels as the 1ng (especially for the R964 - i.e.3'end) while longer PAP times tended to show poorer amplification curves... therefore, 4 min PAP may be sufficient for 10pg RNA quantities
- Its difficult to fully conclude optimal conditions due to the variability... hopefully this is an artifact of the single 10pg beads purification rather than inconsistency/inefficiency of the totoRNAseq reactions at these minimal RNA concentrations