Blue:RNA-Seq Experiments:09102013
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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_ERCC_N2id1_ | ERCC | 1ng - Fragmented | TSO.r04 | T20V.id1 | N2.id1 | totoRNAseq | QC qPCR Analysis | |
BL_ERCC_N2id2_ | ERCC | 1ng - Unfragmented | TSO.R04 | T20V.id1 | N2.id2 | |||
BL_ERCC_N2id3_ | ERCC | 1ng - Fragmented | TSO.R05 | T20V.id1 | N2.id3 | |||
BL_ERCC_N2id4_ | ERCC | 1ng - Unfragmented | TSO.R05 | T20V.id1 | N2.id4 | |||
BL_ERCC_N2id5_ | ERCC | 1ng - Fragmented | Smarter TSO | T20V.id1 | N2.id5 | |||
BL_ERCC_N2id6_ | ERCC | 1ng - Unfragmented | Smarter TSO | T20V.id1 | N2.id6 | |||
BL_ERCC_N2id7_ | ERCC | 1ng - Fragmented | TSO.R04 | Supp.T20.id1 | N2.id7 | |||
BL_ERCC_N2id8_ | ERCC | 1ng - Unfragmented | TSO.R04 | Supp.T20.id1 | N2.id8 |
Purpose
- Examine efficiency of TSO primers using ERCC assay
- Examine efficiency of TSO between fragmented and unfragmented using ERCC assay
Library Preparation (RNAseIII Fragmentation):
- RNA FRAGMENTATION
Volume 1ng RNA 0.5ul 1:1E4 0.2ul 10x RNaseIII Buffer 0.2ul 0.1uM T20V.id1 0.1ul dH20 0.25ul Total 1.25ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- Incubate @ 4C 10min
- RNA FRAGMENTATION
Volume RNA 1.25ul RNase III 0.08ul dH20 0.67ul Total 2ul
- Incubate @ 37C 10 min
- Add 1.1ul dH2O
- Incubate @ 65C 10 min
- 3' Tailing
Volume PNK_fRNA 3.1ul 5x RT Buffer 0.5ul 1mM ATP 0.25ul 0.5U/ul PAP (1:10d in H2O) 0.25ul
- Incubate @ 37C 5 min
- Transfer to Ice
Reverse Transcription:
Volume PAP RNA 4.1ul 10mM dNTP Mix 1ul 0.1uM T20V.ID 0.9ul
- Incubate @ 65C 3 min
- Transfer to Ice
Volume 5xRT Buffer 1.5ul 100mM DTT 0.17ul Betaine (5M) 2ul RNase Inhibitor (40U/ul) 0.1ul Smarter MMLV (200U/ul) 0.23ul 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
TSO
- TSO (x2)
Volume Purified cDNA 3ul 5x RT buffer [MMLV] 2ul 10mM dNTP mix 1ul 100mM DTT 0.25ul 10uM TSO.r04 1ul Betaine (5M) 2ul RNase Inhibitor 0.25ul Smarter MMLV 0.5ul Total 10ul
- Incubate @42C 10min
- Incubate @70C 10min
- USER
- Add 1ul and incubate at 37C for 30 min
- PCR (PhusionHF)
Volume TSO Reaction 11ul 2x Buffer 25ul 10uM PB_PCR_F 1ul 10uM PCR_N2_idX 1ul H2O 12ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 15x 72C, 5min
Results of Amplification
- Ran 1ul on gel:
TSO Quantification
- Comparison of Pre-amp and post-amp plots:
PCR - 5' TSO PCR - 3' T20 PCR fragment TSO+130-5' T20+130-5' ERCC-130-5' TSO+130-mid T20+130-mid ERCC-130-mid TSO+130-3' T20+130-3' ERCC-130-3' TSO+130-5' T20+130-5' ERCC-130-5' TSO+130-mid T20+130-mid ERCC-130-mid TSO+130-3' T20+130-3' ERCC-130-3'
TSO=ILMN_PCR_F; T20=ILMN_PCR_R
- Amplified 1ul TSO and 5ul PCR using Sybr-Green:
Volume Library 1ul or 5ul 2xSybr Buffer 25ul 10uM F primer 1ul 10uM R Primer 1ul dH2O 22ul or 18ul Total 50ul
- 95C 30sec
- 95C 10s -> 60C 20s -> 72C 30s => 5X
- 72C, 5min
- Ran ul on gel: