Blue:RNA-Seq Experiments:totoRNAseq RNaseII Test

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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_ huES8 Single cell TSO.r04 T20V.id1-96 N2.id81-82 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read
huES8 5-10 cells
UHRR 10pg

Plate Layout: Samples and Indexes[edit]

' 1 2 3 4 5 6 7 8 9 10 11 12
A NC(1) UHRR(9) Nc(17) UHRR(25) Nc(33) UHRR(41) Nc(49) UHRR(57) Nc(65) UHRR(73) Nc(81) UHRR(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) 10cells(16) Sc(24) 10cells(32) Sc(40) 10cell(48) Sc(56) 10 cell(64) Sc(72) 10cells(80) Sc(88) 10cells(96)

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

Cells Sorted 08122013


Purpose[edit]

  • Repeat Single cell totoRNAseq protocol last used on 06142013b
    • Test Decrease in T20 concentration from 0.2uM to 0.1uM
    • Test Decrease in total reaction volume

Library Preparation (totoRNAseq):[edit]

Cell Sort
Volume
RNA 10pg or Single Cells -
1x Lysis Buffer 0.5ul
Total 0.5ul


RNA FRAGMENTATION
Volume
RNA 0.5ul
10x RNaseIII Buffer 0.15ul
0.1uM T20V.id 0.1ul
dH20 0.5ul
Total 1.25ul
Incubate @ 72C 3 min
Incubate @ 37C 1min (Centrifuge 1 min during this incubation and add UHRR)
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
PNK_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
dH2O 0ul
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) 0.33ul
Smarter MMLV (200U/ul) 0.5ul
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 (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_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)
  • Resuspend in 50ul
  • Ampure Beads Purify (0.8x)
  • Resuspend in 10ul
  • Run 1ul on gel

Results of Amplification[edit]

  • Ran 1ul on gel:


File:08072013 PCR1 gel1.jpg


N2.id81 = Odd Wells N2.id82 = Even Wells

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[1] 1ul
10uM ILMN_PCR_R[2] 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:08072013 PCR2 gel1.jpg


Re-amplified 0.5ul library using un-modified ILMN_PCR_F/R and ran 5ul of 50 on gel: