Blue:RNA-Seq Experiments:08202013: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''12''' | | align="center" style="background:#f0f0f0;"|'''12''' | ||
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| align="center" style="background:#f0f0f0;"|'''A'''||NC(1)||UHRR(9)||Nc(17)|| | | align="center" style="background:#f0f0f0;"|'''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) | ||
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| align="center" style="background:#f0f0f0;"|'''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) | | align="center" style="background:#f0f0f0;"|'''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) | ||
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| align="center" style="background:#f0f0f0;"|'''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) | | align="center" style="background:#f0f0f0;"|'''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) | ||
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| align="center" style="background:#f0f0f0;"|'''H'''||Sc(8)|| | | align="center" style="background:#f0f0f0;"|'''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) | ||
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Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08122013 08122013] | Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08122013 08122013] | ||
== Purpose == | == Purpose == |
Revision as of 21:07, 19 August 2013
RNA-Seq Library Information
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.id1-48 | N2.id85-86 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read | |
UHRR | 1ng | TSO.R04 | T20V.id1-2 | N2.id 87-88 | ||||
huES8 | Single cell | TSO.r04 | T20V.id49-96 | N2.id87-88 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read | ||
UHRR | 1ng | TSO.R04 | T20V.id1-2 | N2.id 89-90 | ||||
Plate Layout: Samples and Indexes
' | 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
- Repeat Single cell totoRNAseq protocol last used on 06142013b
- Test Decrease in T20 concentration 0.1uM
- Test Decrease in total reaction volume for RT reaction
- Test IVT on single cell and 1ng UHRR
Library Preparation (totoRNAseq):
- 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
- 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:
N2.id81 = Odd Wells
N2.id82 = Even Wells
Final Library Preparation
- Re-Amplified 1ul using Sybr-Green and ILMN_PCR_F/R
- 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:
Re-amplified 0.5ul library using un-modified ILMN_PCR_F/R and ran 5ul of 50 on gel: