Blue:RNA-Seq Experiments:08202013

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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_N2id85_0820 huES8 Single cell TSO.r04 T20V.id1-24 N2.id85 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read
BL_huES8_SingleCell_N2id86_0820 huES8 Single Cell TSO.R04 T20V.id25-48 N2.id86
BL_huES8_SingleCell_N2id87_0820 UHRR 250pg TSO.R04 T20V.id1 N2.id87
BL_huES8_SingleCell_N2id88_0820 UHRR 250pg TSO.R04 T20V.id2 N2.id88
huES8 Single cell RL.IVT.N9 T20V.id49-72 N2.id89
huES8 Single cell RL.IVT.N9 T20V.id73-96 N2.id90
UHRR 250pg RL.IVT.N9 T20V.id1 N2.id91
UHRR 250pg RL.IVT.N9 T20V.id2 N2.id92

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) 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[edit]

  • Repeat Single cell RNAseIII/TSO totoRNAseq protocol last used on 06142013b and compared to 250pg UHRR
    • Test Decrease in T20 concentration 0.1uM
    • Test Decrease in total reaction volume for RT reaction
  • Test single cell RNaseIII/IVT protocol compated to 250pg of UHRR
  • Sort into minimal volume of lysis buffer (0.4ul - bubbled) to keep downstream reaction volumes low

Library Preparation (totoRNAseq):[edit]

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 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) 0.1ul
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[edit]

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


File:08202013 TSO PCR1 gel1.jpg

IVT:[edit]

2nd Strand
Volume
Purified cDNA 7.7ul
FS Buffer 0.2ul
SS Buffer 1ul
dNTP 0.8ul
DNA Pol 0.2ul
RNase H 0.1ul
Total 10ul


Incubate @16C 2hours


Add T7 Promoter
Volume
dsDNA 10ul
KAPA qPCR MM 25ul
RL.Add.T7 (10uM) 0.5ul
dH2O 14.5ul
Total 50ul


Incubate @94C 2min
Incubate @60C 2min
Incubate @72C 5min


Beads Purify
  • Add 50ul beads
  • Wash twice with 80% EtOH
  • Resuspend in 6.4ul


IVT RXN
Volume
dsDNA 6.4ul
ATP 1.6ul
GTP 1.6ul
CTP 1.6ul
UTP 1.6ul
10x T7 Buffer 1.6ul
T7 Polymerase 1.6ul
Total 16ul


Incubate @37C 10 hours; lid 70C
Incubate @4C until ready


RT RXN
Volume
synthesized RNA 5ul
10uM RL.IVT.N9 1ul
Water 1ul
Incubate @65C 5min
Incubate Ice 1min


Volume
10x RT Buffer 1ul
10mM dNTP 0.5ul
100mM DTT 0.5ul
RNase Inh 0.5ul
SSIII 0.5ul
Total 10ul


Incubate @25C 5min
Incubate @50C 20min
Incubate @70C 10min


Beads Purify
  • Add 10ul beads
  • Wash twice with 80% EtOH
  • Resuspend in 23ul dH2O


PCR (PhusionHF) and SYBR
Volume
SSIII Reaction 23ul
2x Buffer 25ul
10uM PB_PCR_F 1ul
10uM PCR_N2_idX 1ul
Total 50ul


98C 30 sec
98C 15s -> 58C 20s -> 72C 30s 6x
98C 10s -> 72C 20s 10x (9x for Sybr)
72C, 5min


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

File:08202013 IVT PCR1 gel1.jpg

Results of Amplification[edit]

  • Ran 1ul on gel:


See "Final Library Preparation" below

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 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:

File:08202013 PCR2 gel1.jpg

  • Re-Amplified 1ul of original id85 and id86 libraries (beads purified 0.8x) and combined with re-amplified libraries shown above
  • Size selection to remove primer dimer band:

File:08202013 size sel gel1.jpg File:08202013 size sel gel2.jpg