Blue:RNA-Seq Experiments:smarter test
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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_UHRR_ | UHRR/ERCC | 1ng - Unfragmented-PolyA | Smarter TSO | SMART CDS | In-house Smarter | Read 1 and N2 barcode read | ||
BL_UHRR_ | UHRR/ERCC | 100pg - Unfragmented-PolyA | Smarter TSO | SMART CDS | In-house Smarter | |||
BL_UHRR_ | UHRR/ERCC | 10pg - Unfragmented-PolyA | Smarter TSO | SMART CDS | In-house Smarter | |||
BL_UHRR_ | UHRR/ERCC | 1ng - Unfragmented | Smarter TSO (Kit) | SMART CDS (Kit) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 100pg - Unfragmented | Smarter TSO (Kit) | SMART CDS (Kit) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 10pg - Unfragmented | Smarter TSO (Kit) | SMART CDS (Kit) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 1ng - RnaseIII Fragmented | Smarter TSO | SMART CDS | In-house Smarter totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 100pg - RNaseIII Fragmented | Smarter TSO | SMART CDS | In-house Smarter totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 10pg - RNaseIII Fragmented | Smarter TSO | SMART CDS | In-house Smarter totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 1ng - RnaseIII Fragmented | Smarter TSO | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 100pg - RNaseIII Fragmented | Smarter TSO | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 10pg - RNaseIII Fragmented | Smarter TSO | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 1ng - RNaseIII Fragmented - 1 step | TSO_r03 | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 100pg - RNaseIII Fragmented - 1 step | TSO_r03 | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 10pg - RNaseIII Fragmented - 1 step | TSO_r03 | T20V.id | N2_id | totoRNAseq |
Purpose
- Examine efficiency of Smarter protocol (unfragmented) compared to Poly-A tailing (unfragmented) and our fragmentation protocol using ERCC assay
- Examine efficiency of the modified TSO r03 (unable to be Poly-A tailed) in a single step totoRNAseq reaction using ERCC assay
Smarter Library Preparation (Unfragmented)
As per Clontech Kit:
- RNA PREPARATION
Volume 2ng/ul, 200ng/ul, 20ng/ul UHRR 0.5ul ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul Dilution Buffer 2.375ul 3' SMART CDS Primer II A 1.0ul RNase Inhibitor 0.125ul Total 4.5ul
- Incubate @ 72C 3 min
Volume 5xRT Buffer 2ul 100mM DTT 0.25ul dNTP (10mM) 1ul Smarter II A Oligonucleotide (12uM) 1ul RNase Inhibitor (40U/ul) 0.25ul Smarter MMLV (200U/ul) 1.0ul Total 10ul
- Incubate @ 42C 90 min
- Incubate @ 70C 10 min
- Beads Purification
- Add 25ul 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 10ul dH2O, incubate 2min, transfer to new tube
- PCR Amplification
Volume cDNA 10ul 2x KAPA HiFi Hotstart ReadyMix 25ul ISPCR primers (10mM) 1.0ul dH2O 14ul Total 50ul
- 98C 3min
- 12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg):
- 98C 15 sec
- 67C 20sec
- 72C 6min
- 72C 5min
Poly-A Unfragmented Library Preparation
- RNA PREPARATION
Volume 2ng/ul, 200ng/ul, 20ng/ul UHRR 0.5ul ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul 10x RNaseIII Buffer 0.2ul 1uM T20V.id1 or CDS II A 0.1ul dH20 0.25ul Total 1.25ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- 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
RNAseIII-Fragmented Library Preparation
- RNA PREPARATION
Volume 2ng/ul, 200ng/ul, 20ng/ul UHRR 0.5ul ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul 10x RNaseIII Buffer 0.2ul 1uM T20V.id1 or CDS II A 0.1ul dH20 0.25ul Total 1.25ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- 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 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
- 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
- Ran 5ul 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=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
- 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: