Blue:RNA-Seq Experiments:01282014
Jump to navigation
Jump to search
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_Jan28 | huNu 1.23.2014 | 1ng sorted pool - 0.1U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id81 | totoRNAseq | ||
huNu 1.23.2014 | 1ng sorted pool - 0.075U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id82 | totoRNAseq | |||
huNu 1.23.2014 | 1ng sorted pool - 0.05U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id83 | totoRNAseq | |||
huNu 1.23.2014 | 1ng sorted pool - 0.025U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id84 | totoRNAseq | |||
huNu 1.23.2014 | 1ng hand-cut - 0.1U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id85 | totoRNAseq | |||
huNu 1.23.2014 | 1ng hand-cut - 0.075U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id86 | totoRNAseq | |||
huNu 1.23.2014 | 1ng hand-cut - 0.05U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id87 | totoRNAseq | |||
huNu 1.23.2014 | 1ng hand-cut - 0.025U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id88 | totoRNAseq | |||
huNu 1.28.2014 | 1ng sorted pool - 0.1U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id89 | totoRNAseq | |||
huNu 1.28.2014 | 1ng sorted pool - 0.075U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id90 | totoRNAseq | |||
huNu 1.28.2014 | 1ng sorted pool - 0.05U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id91 | totoRNAseq | |||
huNu 1.28.2014 | 1ng sorted pool - 0.025U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id92 | totoRNAseq | |||
huNu 1.28.2014 | 1ng hand-cut - 0.1U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id93 | totoRNAseq | |||
huNu 1.28.2014 | 1ng hand-cut - 0.075U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id94 | totoRNAseq | |||
huNu 1.28.2014 | 1ng hand-cut - 0.05U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id95 | totoRNAseq | |||
huNu 1.28.2014 | 1ng hand-cut - 0.025U Rnase3 | TSO.r06 | T20VN_PE_R | N2.id96 | totoRNAseq |
- Primer sequences [1]
- huNu sorted for Fluidigm C1 runs on 01.23.14 and 01.28.14
- RNA was extracted from matched whole brain tissue (area corresponding to huNu) or 100,000 sorted NeuN+ nuclei using Zymo MicroRNA Kit
Purpose[edit]
- To provide pooled RNA-seq libraries of human brain section/sorted nuclei for comparison with Fluidigm C1 single nuclei libraries
RNAseIII-Fragmented Library Preparation[edit]
- RNA
Volume 2ng/ul RNA 0.5ul 1:10E4 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, 0.075ul, 0.05ul, or 0.025ul dH20 0.7ul, 0.725ul, 0.75ul, 0.775ul 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 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, 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) 72C, 5min
Results of Amplification[edit]
- Run 5ul on gel:
Results: 0.1U of RNAseIII (id 81, 85, 89, 93) appears to over fragment this RNA. Anywhere from 0.075 to 0.025 units seems to be effective - sequencing will be needed to determine with condition is best.
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.
- Results: