Blue:RNA-Seq Experiments:08012013

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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_mNPC_SingleCell_0801 mNPC Single cell TSO.r05 T20V.id1-96 N2.id83-84 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read
mNPC 5-10 cells
UHRR 10pg

Plate Layout: Samples and Indexes[edit]

' 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 07102013

Purpose[edit]

  • Optimize Single cell totoRNAseq protocol last used on 07252013
    • Test TSO.R05
    • Increase [Mg] from 9mM to 12mM

Library Preparation (totoRNAseq):[edit]

RNA FRAGMENTATION
Volume
RNA 10pg or Single Cells -
2.5x HiMg RT Buffer/Lysis Buffer[1] 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
Volume
fRNA 2ul
1mM ATP 0.4ul
100mM DTT 0.1ul
Water 0ul
RNase Inhibitor(1:2 or 20U/ul) 0.1ul
PNK 0.4ul
Total 3ul


Incubate @ 37C 30 min


3' Tailing
Volume
PNK_fRNA 3ul
1mM ATP 0.5ul
0.5U/ul PAP (1:10d in H2O) 0.5ul


Incubate @ 37C 5 min
Transfer to Ice


Reverse Transcription
Volume
PAP RNA 4ul
10mM dNTP Mix 1ul
dH2O 0ul
0.2uM T20V.ID 1ul


 Note: accidentally added 'lane 5' into the primers - added 1ul back, but still 1ul short for this lane 
Incubate @ 65C 3 min
Transfer to Ice


Odd Wells Even Wells
5xRT Buffer 1ul 1ul
100mM DTT 0.17ul 0.17ul
Betaine (5M) 2ul 2ul
MgCl2 (300mM) 0ul 0.1ul
RNase Inhibitor (40U/ul) 0.33ul 0.23ul
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 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 (x2)
Volume
Purified cDNA 3ul
5x RT buffer [MMLV] 2ul
10mM dNTP mix 1ul
100mM DTT 0.25ul
10uM TSO.r05 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_Fmod 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) (combined duplicates)
  • Resuspend in 50ul
  • Ampure Beads Purify (0.8x)
  • Resuspend in 10ul
  • Run 1ul on gel

Results of Amplification[edit]

  • Ran 1ul on gel:

File:08012013 PCR1 gel1.jpg


id83=odd wells (9mM Mg2)

id84=even wells (12mM Mg2)

Final Library Preparation[edit]

  • Re-Amplified 1ul using Sybr-Green and ILMN_PCR_F/R
Volume
Library 1ul
2xSybr Buffer 25ul
10uM ILMN_PCR_F[2] 1ul
10uM ILMN_PCR_R[3] 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:

File:08012013 PCR2 gel1.jpgFile:08012013 PCR2.jpg