Blue:RNA-Seq Experiments:11072013

From ZhangLabWiki
Jump to navigation Jump to search

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/ERCC 1ng STRT_v2_3 STRT_V3_T30 Smart-Seq2
BL_ UHRR/ERCC 1ng STRT_v2_3 STRT_V3_T30 Smart-Seq2
BL_ UHRR/ERCC 1ng - Poly(A) STRT_v2_3 STRT_V3_T30 Smart-Seq2
BL_ UHRR/ERCC 1ng - Poly(A) STRT_v2_3 STRT_V3_T30 Smart-Seq2
BL_ UHRR/ERCC 1ng - Poly(A) with dATP STRT_v2_3 STRT_V3_T30 Smart-Seq2
BL_ UHRR/ERCC 1ng - Poly(A) with dATP STRT_v2_3 STRT_V3_T30 Smart-Seq2

Purpose[edit]

  • To test the effectiveness of Smart-Seq2 protocol for partially degraded samples and the potential rescuing effect of PAP

Smart-seq 2 Protocol[edit]

RNA
Volume
UHRR 2ng/ul 0.5ul
ERCC 1:10E4 0.2ul
dH2O 0.3ul
10uM T30 1ul
dNTP (10mM) 1ul
Total 3ul
Incubate @ 72C 3 min
Transfer to ice


Reverse Transcription
Volume
5xRT Buffer 2ul
100mM DTT 0.25ul
100mM MgCl2 0.6ul
Betaine (5M) 2ul
TSO 1ul
dH20 0.4ul
RNase Inhibitor (40U/ul) 0.25ul
Smarter MMLV (200U/ul) 0.5ul
Total 10ul


Incubate @ 42C 90 min
Incubate @ 70C 10 min


Beads Purification
  1. Add 25ul beads per well
  2. Allow to bind 15 min
  3. Remove supernatant and wash 2x with 1ml 80% EtOH
  4. Air Dry until cracking first occurs
  5. Resuspend beads in PCR reaction:


PCR (Advantage)
Volume
10x Advantage 2 PCR Buffer 5ul
dNTP Mix (10mM) 2ul
STRT Primer (10uM) 2ul
50X Advantage 2 Polymerase Mix 2ul
H2O 39ul
Total 50ul


95C 1 min
95C 15s -> 65C 30s -> 68C 6min 12x
72C, 10min

Smart-seq 2 Protocol with Poly(A) Tailing[edit]

RNA
Volume
UHRR 2ng/ul 0.5ul
ERCC 1:10E4 0.2ul
5x ePAP Buffer 0.6ul
dH2O 0.9ul
Total 2.2ul


Incubate @ 72C 3 min
Transfer to ice


Volume
25mM MnCl2 0.3ul
1mM ATP or 1:1 ATP/dATP 0.25ul
ePAP 2u/ul 0.25ul
Total 3ul


Incubate @ 37C 10 min


Volume
10uM T30 1ul
dNTP (10mM) 1ul
Total 5ul


Reverse Transcription
Volume
5xRT Buffer 2ul
100mM DTT 0.25ul
100mM MgCl2 0.3ul
Betaine (5M) 2ul
RNase Inhibitor (40U/ul) 0.25ul
Smarter MMLV (200U/ul) 0.5ul
Total 10.3ul


Incubate @ 42C 90 min


Beads Purification
  1. Add 25ul beads per well
  2. Allow to bind 15 min
  3. Remove supernatant and wash 2x with 1ml 80% EtOH
  4. Air Dry until cracking first occurs
  5. Resuspend beads in TSO reaction:


TSO
Volume
dH2O 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 (Advantage)
Volume
10x Advantage 2 PCR Buffer 5ul
dNTP Mix (10mM) 2ul
STRT Primer (10uM) 2ul
50X Advantage 2 Polymerase Mix 2ul
H2O 29ul
Total 50ul


95C 1 min
95C 15s -> 65C 30s -> 68C 6min 12x
72C, 10min

Results of Amplification[edit]

  • Run 5ul on gel:

File:11072013 PCR1 gel1.jpg


  • Results: Can see that the overall efficiency is abnormally low. We have been having a problem with this and the Smart-Seq protocol lately for some unknown reason. Poly(A) tailing clearly increases the yields of cDNA.

Final Library Preparation[edit]