Blue:RNA-Seq Experiments:07252013

From ZhangLabWiki
Revision as of 22:47, 29 July 2013 by >B1lake (→‎Results of Amplification)
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 N2.id81-82 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 07102013

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 reaction volume - at the same time increasing [Mg] from 8mM to 9mM

Library Preparation (totoRNAseq):

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
Odd Wells Even Wells
fRNA 2ul 2ul
1mM ATP 0.5ul 0.4ul
100mM DTT 0.15ul 0.1ul
Water 1.35ul 0ul
RNase Inhibitor(1:10 or 4U/ul) 0.5ul 0.1ul (1:2 dil)
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
dH2O 1ul 0ul
0.2uM T20V.ID 1ul 1ul


Incubate @ 65C 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 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

  • Ran 1ul on gel:

File:07252013 PCR1 gel1b.jpg


  • Re-Amplified 1ul using Sybr-Green and ILMN_PCR_F/R
  • Ampure Beads purified (0.8x)
  • Resuspended in 10ul and ran 1 ul on gel:

Note: These will be the Pre-RiboZero treated libraries for sequencing

File:07252013 PCR2 gel1.jpgFile:07252013 PCR2.jpg

Ribo-Zero Treatment

Step 1: Re-Amplify to Generate Stable and Unstable DNA Strands


PCR(Sybr)
Volume
Library 5ul
2xSybr Buffer 25ul
10uM ILMN_PCR_F*[2] 1ul
10uM ILMN_PCR_Rb[3] 1ul
dH2O 18ul
Total 50ul


95C 30sec
95C 10s -> 60C 20s -> 72C 30s => 5X
72C, 5min


  • Ampure Beads Purify (0.8x)
  • Resuspend in 12.5ul


Step 2: Exonuclease digestion to generate ssDNA


Lambda Digestion
Volume
Purified PCR Products 12.5ul
10x Lambda Exo Buffer 1.5ul
Lambda Exonuclease 1.0ul
Total 15ul


Incubate 37C 1 hour
  • Ethanol Ppt:
  1. Add 85ul dH2O to bring up to 100ul
  2. Add 0.1x 3M Sodium Acetate (10ul)
  3. Add 2.5x 100%EtoH (250ul)
  4. Add 1ul Glycoblue
  5. Ppt overnight at -80C
  • Resuspend in 11ul
  • Determine concentration using Qubit (ssDNA) on 1ul =


Step 3: rRNA Depletion
Ribo-Zero
  • Thaw components
  • Was magnetic beads and bring to RT:
  • 255ul beads washed 2x with dH2O (equal volume)
  • Resuspend in 65ul (20ul/rxn) resuspension solution
Volume
Ribo-Zero reaction Buffer 1.2ul
Lambda-treated ssDNA (0.25-0.5ug) up to 10ul
Ribo-Zero Gold rRNA removal solution 0.8ul
Total Volume 12ul


  • Vortex beads
  • Add hybridized samples to beads and mix by pipetting 10-15 times and vortex 5 sec
  • Incubate at RT 10min, vortex 5 sec every 3-4 min
  • Vortex 5 sec then heat to 50C 5 min
  • Immediately transfer to magnetic rack and transfer supernatant to new tube
  • Ethanol precipitate as above - resuspend in 10ul


Step 4: Convert to dsDNA


PCR(Sybr)
Volume
Post-Ribo Library 10
2xSybr Buffer 25ul
10uM ILMN_PCR_F 1ul
10uM LIMN_PCR_R 1ul
dH2O 13ul
Total 50ul


95C 30sec
95C 10s -> 60C 20s -> 72C 30s => 4X
72C, 5min


  • Ampure Beads Purify (0.8x)
  • Resuspend in 10ul