Blue:RNA-Seq Experiments:10012013

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Revision as of 22:18, 1 October 2013 by >B1lake
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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_huES8_SingleCell_N2id_ huES8 Single cell TSO.r06 T20V.id1-24 N2.id85 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read

Plate Layout: Samples and Indexes

' 1 2 3 4 5 6 7 8 9 10 11 12
A NC(1) UHRR(9) Nc(17) Nc(25) UHRR(33) NC(41) Nc(49) UHRR(57) Nc(65) Nc(73) UHRR(81) NC(89)
B Sc(2) Sc(10) Sc(18) Sc(26) Sc(34) Sc(42) Sc(50) Sc(58) Sc(66) Sc(74) Sc(82) Sc(90)
C Sc(3) Sc(11) Sc(19) Sc(27) Sc(35) Sc(43) Sc(51) Sc(59) Sc(67) Sc(75) Sc(83) Sc(91)
D Sc(4) Sc(12) Sc(20) Sc(28) Sc(36) Sc(44) Sc(52) Sc(60) Sc(68) Sc(76) Sc(84) Sc(92)
E Sc(5) Sc(13) Sc(21) Sc(29) Sc(37) Sc(45) Sc(53) Sc(61) Sc(69) Sc(77) Sc(85) Sc(93)
F Sc(6) Sc(14) Sc(22) Sc(30) Sc(38) Sc(46) Sc(54) Sc(62) Sc(70) Sc(78) Sc(86) Sc(94)
G Sc(7) Sc(15) Sc(23) Sc(31) Sc(39) Sc(47) Sc(55) Sc(63) Sc(71) Sc(79) Sc(87) Sc(95)
H Sc(8) Sc(16) 10cells(24) Sc(32) Sc(40) 10cell(48) Sc(56) Sc(64) 10cells(72) Sc(80) Sc(88) 10cells(96)

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

Cells Sorted 08122013


Purpose

  • Generate totoRNAseq libraries using modified Smarter TSO (r06) and modified r04 TSO to compare efficiencies
  • Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal RNA generated from sorted nuclei or LCM
  • Compare RNAseIII fragmentation times: full (10min) or partial (5min)


RNAseIII-Fragmented Library Preparation

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

Note: add in UHRR control at this point


RNA FRAGMENTATION
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 5-10 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
  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 - vacuspin 5 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 (Smarter TSO) or PB_PCR_F 1ul
10uM PCR_R_N2_id 1ul
H2O 12ul
Total 50ul


98C 30 sec
98C 15s -> 58C 20s -> 72C 30s 6x
98C 10s -> 72C 20s 12x
72C, 5min

Results of Amplification

  • Run 5ul on gel:


Final Library Preparation