Blue:RNA-Seq Experiments:07252013: Difference between revisions
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|PNK||||||||||0.5ul||||||||||0.4ul | |PNK||||||||||0.5ul||||||||||0.4ul | ||
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|Total||||||||||5ul|||||||||| | |Total||||||||||5ul||||||||||3ul | ||
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:::''Incubate @ 37C 30 min'' | :::''Incubate @ 37C 30 min'' | ||
:'''3' Tailing''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Odd Wells</u>||||||||||<u>Even Wells</u> | |||
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|PNK_fRNA||||||||||5ul||||||||||3ul | |||
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|1mM ATP||||||||||0.5ul||||||||||0.5ul | |||
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|0.5U/ul PAP (1:10d in H2O)||||||||||0.5ul||||||||||0.5ul | |||
|- | |||
|} | |||
:::''Incubate @ 37C 5 min'' | |||
:::''Transfer to Ice'' | |||
:'''Reverse Transcription''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Odd Wells</u>||||||||||<u>Even Wells</u> | |||
|- | |||
|PAP RNA||||||||||6ul||||||||||4ul | |||
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|10mM dNTP Mix||||||||||1ul||||||||||1ul | |||
|- | |||
|0.1uM T20V.ID||||||||||1ul||||||||||1ul | |||
|- | |||
|} | |||
:::''Incubate @ 70C 3 min'' | |||
:::''Transfer to Ice'' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Odd Wells</u>||||||||||<u>Even Wells</u> | |||
|- | |||
|5xRT Buffer||||||||||2ul|||||||||| ul | |||
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|100mM DTT||||||||||0.25ul|||||||||| ul | |||
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|Betaine (5M)||||||||||3ul|||||||||| ul | |||
|- | |||
|RNase Inhibitor||||||||||0.25ul||||||||||0.5ul | |||
|- | |||
|Total||||||||||15ul||||||||||10ul | |||
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|} |
Revision as of 21:50, 24 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 | 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 06182013[1]
Purpose
- Optimize Single cell totoRNAseq protocol last used on 06142013 (and 06142013b) - try to decrease primer dimers
- Reduce T20 primer concentration
- Reduce betaine volume
- Test R05
Library Preparation (totoRNAseq):
- RNA FRAGMENTATION
Volume RNA 10pg or Single Cells - 2.5x HiMg RT Buffer/Lysis Buffer[2] 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 0.35ul 100mM DTT 0.15ul 0.15ul Water 1.35ul 0ul RNase Inhibitor(1:10 or 4U/ul) 0.5ul 0.1ul (undil) 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 0.1uM T20V.ID 1ul 1ul
- Incubate @ 70C 3 min
- Transfer to Ice
Odd Wells Even Wells 5xRT Buffer 2ul ul 100mM DTT 0.25ul ul Betaine (5M) 3ul ul RNase Inhibitor 0.25ul 0.5ul Total 15ul 10ul