Blue:RNA-Seq Experiments:07252013: Difference between revisions
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:::*Ampure Beads Purify (0.8x) | :::*Ampure Beads Purify (0.8x) | ||
:::*Resuspend in 12.5ul | :::*Resuspend in 12.5ul | ||
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:::*Resuspend in 11ul | :::*Resuspend in 11ul | ||
:::*Determine concentration using Qubit (ssDNA) on 1ul = | :::*Determine concentration using Qubit (ssDNA) on 1ul = | ||
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:::*Immediately transfer to magnetic rack and transfer supernatant to new tube | :::*Immediately transfer to magnetic rack and transfer supernatant to new tube | ||
:::*Ethanol precipitate as above - resuspend in 10ul | :::*Ethanol precipitate as above - resuspend in 10ul | ||
:'''Step 4: Convert to dsDNA''' | :'''Step 4: Convert to dsDNA''' |
Revision as of 23:50, 26 July 2013
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
- Add 15ul beads per well
- Allow to bind 15 min
- Combine all odd or even column wells
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 20ul dH2O, incubate 2min, transfer to new tube
- 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:
- 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:
File:07252013 PCR2 gel1.jpgFile:07252013 PCR2.jpg
Ribo-Zero Treatment
- Step 1: Re-Amplify to Generate Stable and Unstable DNA Strands
- PCR(Sybr)
- 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:
- Add 85ul dH2O to bring up to 100ul
- Add 0.1x 3M Sodium Acetate (10ul)
- Add 2.5x 100%EtoH (250ul)
- Add 1ul Glycoblue
- 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