Blue:RNA-Seq Experiments:08282013

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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_HSC_SingleCell_N2id89_0828 huES8 Single cell TSO.r04 T20V.id1-24 N2.id89 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read
UHRR 250pg TSO.r04 T20V.id1 N2.id90
UHRR 250pg TSO.r04 T20V.id2 N2.id91

Plate Layout: Samples and Indexes

' 1 2 3
A 10cell(1) 10cell(9) Sc(17)
B Sc(2) Sc(10) Sc(18) Sc(26)
C Sc(3) Sc(11) Sc(19) Sc(27)
D Sc(4) Sc(12) Sc(20) Sc(28)
E Sc(5) Sc(13) Sc(21) Sc(29)
F Sc(6) Sc(14) Sc(22) Sc(30)
G Sc(7) Sc(15) Sc(23) Sc(31)
H Sc(8) Sc(16) 10cells(24) Sc(32)

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

Cells Sorted

Purpose

  • Try Single cell RNAseIII/TSO totoRNAseq protocol on Zhili's HSC sorted cells
    • Cells are in hiMg based buffer, however RNaseIII protocol looks more promising (08202013)
    • Test RNaseIII adaptation using hiMg RT sort buffer

Library Preparation (totoRNAseq):

Cell Sort
Volume
RNA 250pg or Single Cells -
2.5x hiMgRT/Lysis Buffer 2ul
Total 2ul

Note: add in UHRR control at this point


RNA FRAGMENTATION
Volume
RNA 2ul
0.1uM T20V.id 0.1ul
dH20 0.45ul
Total 2.55ul
Incubate @ 72C 3 min
Incubate @ 37C 1min
Incubate @ 4C 10min


RNA FRAGMENTATION
Volume
RNA 2.55ul
RNase III 0.08ul
dH20 0.47ul
Total 3.1ul


Incubate @ 37C 10 min
Incubate @ 65C 10 min


3' Tailing
Volume
PNK_fRNA 3.1ul
dH2O 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
dH2O 0.5ul
0.1uM T20V.ID 0.9ul


Incubate @ 65C 3 min
Transfer to Ice


Volume
5xRT Buffer 1ul
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. Combine all odd or even column wells
  4. Remove supernatant and wash 2x with 1ml 80% EtOH
  5. Air Dry 10min or until cracking first occurs
  6. Resuspend beads in 20ul dH2O, incubate 2min, transfer to new tube
  7. Vacuspin room temperature for 10+min to bring volume to 6ul (TSO) or 7.7ul (IVT)


TSO

TSO (x2)
Volume
Purified cDNA 3ul
5x RT buffer [MMLV] 2ul
10mM dNTP mix 1ul
100mM DTT 0.25ul
10uM TSO.r04 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 1ul
10uM PCR_N2_idX 1ul
H2O 12ul
Total 50ul


98C 30 sec
98C 15s -> 58C 20s -> 72C 30s 6x
98C 10s -> 72C 20s 15x
72C, 5min


  • Ampure Beads Purify (0.8x)
  • Resuspend in 50ul
  • Ampure Beads Purify (0.8x)
  • Resuspend in 10ul
  • Run 1ul on gel


Results of Amplification

  • Ran 1ul on gel:


Final Library Preparation

  • Re-Amplified 1ul using Sybr-Green and ILMN_PCR_F/R
Volume
Library 1ul
2xSybr Buffer 25ul
10uM ILMN_PCR_F 1ul
10uM ILMN_PCR_R 1ul
dH2O 22ul
Total 50ul


95C 30sec
95C 10s -> 60C 20s -> 72C 30s => 5X
72C, 5min


  • Ampure Beads purified (0.8x)
  • Resuspended in 10ul and ran 1 ul on gel: