Blue:RNA-Seq Experiments:07252013

From ZhangLabWiki
Revision as of 23:07, 24 July 2013 by >B1lake (→‎Purpose)
Jump to navigation Jump to search

RNA-Seq Library Information

Library ID Samples Input RNA TSO Primer Bar Code Primers Bar Code Primer 2 Seq Primer Type of Seq Reads Library Conc
BL_mNPC_SingleCell_0725 mNPC Single cell TSO.R04 T20V.id1-96 totoRNAseq Read 1 plus N2 barcode read
mNPC 5-10 cells
UHRR 10pg


Plate Layout: Samples and Indexes

' 1 2 3 4 5 6 7 8 9 10 11 12
A Sc(1) Sc(9) Sc(17) Sc(25) Sc(33) Sc(41) Sc(49) Sc(57) Sc(65) Sc(73) Sc(81) Sc(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) Sc(24) Sc(32) 5 cell(40) 5 cell(48) 10 cell(56) 10 cell(64) UHRR(72) UHRR(80) Nc(88) Nc(96)

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

Cells Sorted 06182013[1]

Purpose

  • Optimize Single cell totoRNAseq protocol last used on 06142013 (and 06142013b)
  • Reduce T20 primer concentration - try to decrease primer dimers/adaptor only reads
  • Reduce global volume

Library Preparation (totoRNAseq):

RNA FRAGMENTATION
Volume
RNA 10pg or Single Cells -
2.5x HiMg RT Buffer/Lysis Buffer[2] 2ul
Total 2ul


Incubate @ 72C 3 min
Incubate @ 4C 10min (Centrifuge 1 min during this incubation and add UHRR)
Incubate @ 25C 1min
Incubate @ 94C 2 min
Transfer to ice


End Repair
Odd Wells Even Wells
fRNA 2ul 2ul
1mM ATP 0.5ul 0.35ul
100mM DTT 0.15ul 0.15ul
Water 1.35ul 0ul
RNase Inhibitor(1:10 or 4U/ul) 0.5ul 0.1ul (undil)
PNK 0.5ul 0.4ul
Total 5ul 3ul


Incubate @ 37C 30 min


3' Tailing
Odd Wells Even Wells
PNK_fRNA 5ul 3ul
1mM ATP 0.5ul 0.5ul
0.5U/ul PAP (1:10d in H2O) 0.5ul 0.5ul


Incubate @ 37C 5 min
Transfer to Ice


Reverse Transcription
Odd Wells Even Wells
PAP RNA 6ul 4ul
10mM dNTP Mix 1ul 1ul
0.1uM T20V.ID 1ul 1ul


Incubate @ 70C 3 min
Transfer to Ice


Odd Wells Even Wells
5xRT Buffer 2ul 1ul
100mM DTT 0.25ul 0.17ul
Betaine (5M) 3ul 2ul
RNase Inhibitor (40U/ul) 0.25ul 0.33ul
Smarter MMLV (200U/ul) 0.5ul 0.5ul Total 15ul 10ul


Incubate @ 42C 30 min


Beads Purification
  1. Add 15ul beads per well
  2. Allow to bind 15 min
  3. Combine all 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