Blue:RNA-Seq Experiments:10012013

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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_huES8_SingleCell_N2id_89 huES8 Single cell TSO.r06 T20V.id1-24 (no 17) N2.id89 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read
BL_huES8_SingleCell_N2id_90 UHRR 250pg TSO.r06 T20V.id17 N2.id90 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read

Plate Layout: Samples and Indexes[edit]

' 1 2 3 4 5 6 7 8 9 10 11 12
A NC(1) UHRR 10pg(9) UHRR 250pg(17)
B Sc(2) Sc(10) Sc(18)
C Sc(3) Sc(11) Sc(19)
D Sc(4) Sc(12) Sc(20)
E Sc(5) Sc(13) Sc(21)
F Sc(6) Sc(14) Sc(22)
G Sc(7) Sc(15) Sc(23)
H Sc(8) Sc(16) 10cells(24)

Note: SC=Single cell; UHRR=Universal Human Reference RNA

Cells Sorted 08122013

Purpose[edit]

  • Generate totoRNAseq single cell libraries using modified Smarter TSO (r06) as done in 08202013 that involved r04
  • Compare r04 with r06

RNAseIII-Fragmented Library Preparation[edit]

Cell Sort
Volume
RNA 250pg or Single Cells -
1x Lysis Buffer 0.4ul
Total 0.4ul

Note: add in UHRR control at this point


Lysis
Volume
RNA 0.4ul
10x RNaseIII Buffer 0.2ul
0.1uM T20V.id 0.1ul
dH20 0.55ul
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
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
  1. Add 15ul beads per well
  2. Allow to bind 15 min, combine
  3. Remove supernatant and wash 2x with 1ml 80% EtOH
  4. Air Dry 10min or until cracking first occurs
  5. Resuspend beads in 10ul dH2O, incubate 2min, transfer to new tube - vacuspin 8 min to bring to 3ul


TSO
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 P1-STRT 1ul
10uM PCR_R_N2_id89,90 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]

  • Run 5ul on gel:

File:10012013 PCR1 gel1.jpg

Final Library Preparation[edit]

  • Beads purify (0.8x), re-suspend in 10ul
  • Run 0.5ul on gel:

File:10012013 PCR1 gel2.jpg


  • PCR of 0.5ul using ILMN_PCR_F/R:

File:10012013 PCR2 PCRF-R.jpg


  • PCR of 0.5ul using ILMN_PCR_F/ERCC130_R227:

File:10012013 PCR2 ERCC.jpg


Note:

  • Pre-PCR TSO control is RnaseIII Smarter TSO control from 09102013
  • To add both 10pg and 250pg UHRR to the plate, lysis buffer (containing ERCC) was removed and 0.4ul UHRR was added - the ERCC expression for id90 should be much smaller than id89 (which has 23 combined wells...)