Blue:RNA-Seq Experiments:09102013
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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_UHRR_N2id9_0910 | UHRR/ERCC | 0.5ng - RNaseIII Fragmented | TSO.r04 | T20V.id96 | N2_id9 | totoRNAseq | QC Analysis | |
BL_UHRR_N2id10_0910 | UHRR/ERCC | 0.5ng - Mg Fragmented | TSO.R04 | T20V.id96 | N2_id10 | |||
UHRR/ERCC | 0.5ng - Unfragmented | TSO.R04 | T20V.id96 | N/A | ||||
BL_UHRR_N2id11_0910 | UHRR/ERCC | 0.5ng - RNaseIII Fragmented | TSO.R05 | T20V.id96 | N2_id11 | |||
BL_UHRR_N2id12_0910 | UHRR/ERCC | 0.5ng - Mg Fragmented | TSO.R05 | T20V.id96 | N2_id12 | |||
UHRR/ERCC | 0.5ng - Unfragmented | TSO.R05 | T20V.id96 | N/A | ||||
BL_UHRR_N2id13_0910 | UHRR/ERCC | 0.5ng - RNAseIII Fragmented | Smarter TSO | T20V.id96 | N2_id13 | |||
BL_UHRR_N2id14_0910 | UHRR/ERCC | 0.5ng - Mg Fragmented | Smarter TSO | T20V.id96 | N2_id14 | |||
UHRR/ERCC | 0.5ng - Unfragmented | Smarter TSO | T20V.id96 | N/A | ||||
UHRR/ERCC | 0.5ng - RNaseIII Fragmented | TSO.R04 | Supp.T20.id1 | N/A | ||||
UHRR/ERCC | 0.5ng - Mg Fragmented | TSO.R04 | Supp.T20.id1 | N/A | ||||
UHRR/ERCC | 0.5ng - Unfragmented | TSO.R04 | Supp.T20.id1 | N/A |
Purpose[edit]
- Examine efficiency of our TSO primers (r04/r05) compared to Smarter TSO using ERCC assay
- Examine efficiency of each TSO between fragmented and unfragmented using ERCC assay
- Compare RNaseIII and Mg based fragmentation methods
Library Preparation (RNAseIII Fragmentation):[edit]
- RNA FRAGMENTATION
Volume 1ng/ul UHRR 0.5ul ERCC 1:10E4 0.2ul 10x RNaseIII Buffer 0.2ul 1uM T20V.id1 or Supp.T20.id 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
Library Preparation (Mg-based Fragmentation)[edit]
- RNA FRAGMENTATION
Volume 1ng/ul UHRR RNA 0.5ul 1:1E104 ERCC 0.2ul 5x RT buffer 1ul dH2O 0.3ul Total 2ul
- Incubate @ 94C 2 min
- Transfer to ice
- End Repair
Volume fRNA 2ul 1mM ATP 0.4ul 100mM DTT 0.1ul RNase Inhibitor 0.1ul PNK 0.5ul Total 3.1ul
- Incubate @ 37C 30 min
- 3' Tailing
Volume PNK_fRNA 3.1ul 1mM ATP 0.5ul 0.5U/ul PAP (1:10d in H2O) 0.5ul
- Incubate @ 37C 5 min
- Transfer to Ice
Reverse Transcription:[edit]
Volume PAP RNA or unfrag RNA (diluted to 3.6ul in dH2O) 4.1ul (3.6ul) 10mM dNTP Mix 1ul 1uM T20V.ID or Supp.T20.id 0.9ul
- Incubate @ 65C 3 min
- Transfer to Ice
Volume 5xRT Buffer 1.5ul (RNaseIII); 1ul + 0.5ul dH2O (Mg); 2ul (unfrag) 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[edit]
- TSO (x2)
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
- USER
- Add 1ul and incubate at 37C for 30 min
- PCR (PhusionHF)
Volume TSO Reaction 11ul 2x Buffer 25ul 10uM PB_PCR_F; P1-STRT (Smarter TSO); or PCR_Blue_Short 1ul 10uM PGR_PCR_R or PCR_Blue_Short 1ul H2O 12ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 11x 72C, 5min
Results of Amplification[edit]
- Ran 5ul on gel:
TSO Quantification[edit]
- 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=ILMN_PCR_F; T20=ILMN_PCR_R
- Amplified 1ul TSO (diluted 5ul to 50ul) and 1ul PCR (total volume 50ul) using Sybr-Green:
Volume Library 1ul 2xSybr Buffer 12.5ul 10uM F primer 0.5ul 10uM R Primer 0.5ul dH2O 10.5ul Total 25ul
- 95C 30sec
- 95C 10s -> 60C 20s -> 72C 30s => 40X
- 72C, 5min
Loading Format for qPCR:
' | ' | RnaseIII | ' | ' | Mg | ' | ' | Unfrag | ' |
5' | mid | 3' | 5' | mid | 3' | 5' | mid | 3' | |
TSO | r04 | r04 | r04 | r04 | r04 | r04 | r04 | r04 | r04 |
TSO | r05 | r05 | r05 | r05 | r05 | r05 | r05 | r05 | r05 |
TSO | Smart | Smart | Smart | Smart | Smart | Smart | Smart | Smart | Smart |
TSO | r04+Supp | r04+Supp | r04+Supp | r04+Supp | r04+Supp | r04+Supp | r04+Supp | r04+Supp | r04+Supp |
PCR | r04 | r04 | r04 | r04 | r04 | r04 | r04 | r04 | r04 |
PCR | r05 | r05 | r05 | r05 | r05 | r05 | r05 | r05 | r05 |
PCR | Smart | Smart | Smart | Smart | Smart | Smart | Smart | Smart | Smart |
PCR | r04+Supp | r04+Supp | r04+Supp | r04+Supp | r04+Supp | r04+Supp | r04+Supp | r04+Supp | r04+Supp |
File:09102013 ERCC-R PCR-F.jpg
File:09102013 ERCC130 Unfrag.jpg
File:09102013 ERCC130 RIII.jpg
Final Library Preparation[edit]
- Beads Purified PCR product (0.8x) and resuspended in 10ul:
- Re-Amplified using PB_PCR_F or P1-STRT and PCR_R_N2_id primers:
- r04 - RnaseIII - id9
- r04 - Mg - id10
- r05 - RnaseIII - id11
- r05 - Mg - id12
- Smart - RnaseIII - id13
- Smart - Mg - id14
Volume Library 1ul Smart or 4ul r04/r05 2xSybr Buffer 25ul 10uM F primer 1ul 10uM R Primer 1ul dH2O up to 50ul
- 95C 30sec
- 95C 10s -> 60C 20s -> 72C 30s => 5X
- 72C, 5min
- Beads purified (0.8x), resuspended in 10ul and ran 1ul on gel: