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
  1. Add 15ul beads per well
  2. Allow to bind 15 min
  3. Remove supernatant and wash 2x with 1ml 80% EtOH
  4. Air Dry 10min or until cracking first occurs
  5. 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:

File:09102013 PCR1 gel1.jpg

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

File:09102013 ERCC130 Mg.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:
  1. r04 - RnaseIII - id9
  2. r04 - Mg - id10
  3. r05 - RnaseIII - id11
  4. r05 - Mg - id12
  5. Smart - RnaseIII - id13
  6. 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:

File:09102013 PCR2 gel1.jpg