Blue:RNA-Seq Experiments:01222014
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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_Jan22 | UHRR/ERCC | 10pg - 0.1 RNase3 1min/Affymetrix PAP 4min | TSO.r06 | T20VN_id1-10 | N2.id81 | totoRNAseq | ||
UHRR/ERCC | 10pg - 0.05 RNase3 1min/Affymetrix PAP 4min | TSO.r06 | T20VN_id1-10 | N2.id82 | totoRNAseq | |||
UHRR/ERCC | 10pg - 0.01 RNase3 1min/Affymetrix PAP 4min | TSO.r06 | T20VN_id1-10 | N2.id83 | totoRNAseq | |||
UHRR/ERCC | 1ng - 0.1 RNase3 1min/Affymetrix PAP 10min | TSO.r06 | T20VN_id1 | N2.id84-87 | totoRNAseq | |||
UHRR/ERCC | 100pg - 0.1 RNase3 1min/Affymetrix PAP 8min | TSO.r06 | T20VN_id1 | N2.id88-91 | totoRNAseq |
- Note: 10pg/0.01U T20VN.id1 was actually 0.1U RNase3
Primer sequences [1]
Purpose[edit]
- Given the difficulty in concluding optimal conditions from individual 10pg libraries, will test different RNaseIII treatment conditions on combined 10pg UHRR libraries
- Previous RNaseIII optimizations involved reducing the time for this reaction down to 15s, but this is quite short and might still not be optimal. Will keep the reaction time constant and try reducing the [RNaseIII]
- 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 ([2])
RNAseIII-Fragmented Library Preparation[edit]
- RNA
Volume 2ng/ul or 0.02ng/ul UHRR 0.5ul 1:10E4 or 1:10E6 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, 0.05ul (+ 0.05ul dH2O), or 0.1ul of 1:10 dil dH20 0.7ul Total 2ul
- Incubate @ 37C 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 4, 8 or 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 @ *37C 30 min + 30 min
- accidentally incubated at 37C then extended reaction to be safe
- 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 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[edit]
- Run 5ul on gel:
Results: Similar smear amplification for all conditions
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:01222014 ERCC130 R227.jpgFile:01222014 ERCC130 R964.jpg
- Results: ERCC amplication for the combined 10pg libraries was more similar to the 1ng in this experiment. 100pg libraries showed poorer amplification (not shown) in comparison - might be due to the reduction of PAP from 10 min to 8 min - this may not be optimal. Comparing the different amounts of RNase3 (0.1-0.01U), we can see that the 0.1U actually showed similar amplifications to the 1ng replicates, while 0.01U units was less effective. Interestingly, the 0.05U actually seemed to be improved over the 1ng condition. Therefore, 1 minute incubation with 0.05U RNase3 might be the best condition for 10pg. Also, its possible that the 1 hour RT reaction at 37C improved the outcome for these lower inputs. Therefore, will need to generate similar libraries at 42C for comparison.