Blue:RNA-Seq Experiments:01232014
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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 | huNu | 500 nuclei | TSO.r06 | T20VN_PE_R | N2.id1-2 | totoRNAseq | ||
huNu | 1ng sorted pool | TSO.r06 | T20VN_PE_R | N2.id3-4 | totoRNAseq | |||
huNu | 1ng hand-cut | TSO.r06 | T20VN_PE_R | N2.id5-6 | totoRNAseq |
- Primer sequences [1]
- huNu sorted for Fluidigm C1 run on 01232014
- RNA was extracted from whole brain tissue 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 huNu RNA or 1000 nuclei 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 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 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: Amplification did not work for 500 nuclei - likely they did not lyse properly. Sorted huNu and whole brain tissue samples amplified well, but might be over-fragmented (smear appears to be shifted down). Will repeat totoRNAseq using lower concentrations of RNase to reduce extent of fragmentation.
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: