Blue:RNA-Seq Experiments:10012013: Difference between revisions
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>B1lake (Created page with " == RNA-Seq Library Information == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Samples''' | al...") |
>B1lake No edit summary |
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| align="center" style="background:#f0f0f0;"|'''TSO Primer''' | | align="center" style="background:#f0f0f0;"|'''TSO Primer''' | ||
| align="center" style="background:#f0f0f0;"|'''Bar Code Primers''' | | align="center" style="background:#f0f0f0;"|'''Bar Code Primers''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''Bar Code Primer 2''' | ||
| align="center" style="background:#f0f0f0;"|'''Type of Seq''' | | align="center" style="background:#f0f0f0;"|'''Type of Seq''' | ||
| align="center" style="background:#f0f0f0;"|'''Reads''' | | align="center" style="background:#f0f0f0;"|'''Reads''' | ||
| align="center" style="background:#f0f0f0;"|'''Library Conc''' | | align="center" style="background:#f0f0f0;"|'''Library Conc''' | ||
|- | |- | ||
| | | BL_huES8_SingleCell_N2id_||huES8||Single cell||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id1-24]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id85]||totoRNAseq||Read1 plus N2 barcode read and Read2 for T20 barcode read|| | ||
|- | |- | ||
| | |} | ||
| | |||
| | == Plate Layout: Samples and Indexes == | ||
| | {| {{table}} | ||
| | | align="center" style="background:#f0f0f0;"|'''''' | ||
| align="center" style="background:#f0f0f0;"|'''1''' | |||
| align="center" style="background:#f0f0f0;"|'''2''' | |||
| align="center" style="background:#f0f0f0;"|'''3''' | |||
| align="center" style="background:#f0f0f0;"|'''4''' | |||
| align="center" style="background:#f0f0f0;"|'''5''' | |||
| align="center" style="background:#f0f0f0;"|'''6''' | |||
| align="center" style="background:#f0f0f0;"|'''7''' | |||
| align="center" style="background:#f0f0f0;"|'''8''' | |||
| align="center" style="background:#f0f0f0;"|'''9''' | |||
| align="center" style="background:#f0f0f0;"|'''10''' | |||
| align="center" style="background:#f0f0f0;"|'''11''' | |||
| align="center" style="background:#f0f0f0;"|'''12''' | |||
|- | |- | ||
| | | 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) | ||
|- | |- | ||
| | | 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;"|'''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) | ||
|- | |- | ||
| | | align="center" style="background:#f0f0f0;"|'''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) | ||
|- | |- | ||
| | | align="center" style="background:#f0f0f0;"|'''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) | ||
|- | |- | ||
| | | align="center" style="background:#f0f0f0;"|'''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) | ||
|- | |- | ||
| | | 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;"|'''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 | |||
[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue: | Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08122013 08122013] | ||
== Purpose == | == Purpose == | ||
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*Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal RNA generated from sorted nuclei or LCM | *Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal RNA generated from sorted nuclei or LCM | ||
*Compare RNAseIII fragmentation times: full (10min) or partial (5min) | *Compare RNAseIII fragmentation times: full (10min) or partial (5min) | ||
== RNAseIII-Fragmented Library Preparation == | == RNAseIII-Fragmented Library Preparation == | ||
:''' | :'''Cell Sort''' | ||
::{| {{table}} | ::{| {{table}} | ||
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|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |RNA 250pg or Single Cells||||||||||- | ||
|- | |||
|1x Lysis Buffer||||||||||0.4ul | |||
|- | |- | ||
| | |Total||||||||||0.4ul | ||
|- | |||
|} | |||
''Note: add in UHRR control at this point'' | |||
:'''RNA FRAGMENTATION''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|RNA||||||||||0.4ul | |||
|- | |- | ||
|10x RNaseIII Buffer||||||||||0.2ul | |10x RNaseIII Buffer||||||||||0.2ul | ||
|- | |- | ||
| | |0.1uM T20V.id||||||||||0.1ul | ||
|- | |- | ||
|dH20||||||||||0. | |dH20||||||||||0.55ul | ||
|- | |- | ||
|Total||||||||||1.25ul | |Total||||||||||1.25ul | ||
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:::''Incubate @ 72C 3 min'' | :::''Incubate @ 72C 3 min'' | ||
:::''Incubate @ 37C 1min'' | :::''Incubate @ 37C 1min'' | ||
:::''Incubate @ 4C 10min'' | |||
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|10mM dNTP Mix||||||||||1ul | |10mM dNTP Mix||||||||||1ul | ||
|- | |- | ||
| | |0.1uM T20V.ID ||||||||||0.9ul | ||
|- | |- | ||
|} | |} | ||
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|98C 15s -> 58C 20s -> 72C 30s||||||||||6x | |98C 15s -> 58C 20s -> 72C 30s||||||||||6x | ||
|- | |- | ||
|98C 10s -> 72C 20s|||||||||| | |98C 10s -> 72C 20s||||||||||12x | ||
|- | |- | ||
|72C, 5min | |72C, 5min | ||
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*Run 5ul on gel: | *Run 5ul on gel: | ||
== Final Library Preparation == | == Final Library Preparation == |
Revision as of 22:18, 1 October 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_N2id_ | huES8 | Single cell | TSO.r06 | T20V.id1-24 | N2.id85 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read |
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
- Generate totoRNAseq libraries using modified Smarter TSO (r06) and modified r04 TSO to compare efficiencies
- Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal RNA generated from sorted nuclei or LCM
- Compare RNAseIII fragmentation times: full (10min) or partial (5min)
RNAseIII-Fragmented Library Preparation
- 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 5-10 min
- Add 1.1ul dH2O
- Incubate @ 65C 10 min
- 3' Tailing
Volume 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 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
- Add 15ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube - vacuspin 5 min to bring to 3ul
- TSO
Volume Purified cDNA 3ul 5x RT buffer [MMLV] 2ul 10mM dNTP mix 1ul 100mM DTT 0.25ul 10uM TSO 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 P1-STRT (Smarter TSO) or PB_PCR_F 1ul 10uM PCR_R_N2_id 1ul H2O 12ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 12x 72C, 5min
Results of Amplification
- Run 5ul on gel: