Blue:RNA-Seq Experiments:07252013: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''Bar Code Primers''' | | align="center" style="background:#f0f0f0;"|'''Bar Code Primers''' | ||
| align="center" style="background:#f0f0f0;"|'''Bar Code Primer 2''' | | 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_mNPC_SingleCell_0725||mNPC||Single cell||TSO. | | BL_mNPC_SingleCell_0725||mNPC||Single cell||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r04]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id1-96]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id81-82]||totoRNAseq||Read1 plus N2 barcode read and Read2 for T20 barcode read|| | ||
|- | |- | ||
| ||mNPC||5-10 cells | | ||mNPC||5-10 cells|||||||||||| | ||
|- | |- | ||
| ||UHRR||10pg | | ||UHRR||10pg|||||||||||| | ||
|- | |- | ||
| | | | ||
|} | |} | ||
== Plate Layout: Samples and Indexes == | == Plate Layout: Samples and Indexes == | ||
Line 42: | Line 40: | ||
| align="center" style="background:#f0f0f0;"|'''12''' | | align="center" style="background:#f0f0f0;"|'''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) | | align="center" style="background:#f0f0f0;"|'''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) | | 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) | ||
|- | |- | ||
| 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;"|'''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) | | 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) | ||
|- | |- | ||
| 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;"|'''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) | | 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) | ||
|- | |- | ||
| 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) | ||
|- | |- | ||
| 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) | | align="center" style="background:#f0f0f0;"|'''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) | ||
|- | |- | ||
| | | | ||
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Note: SC=Single cell; UHRR=Universal Human Reference RNA | Note: SC=Single cell; UHRR=Universal Human Reference RNA | ||
Cells Sorted | Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:07102013 07102013] | ||
== Purpose == | == Purpose == | ||
*Optimize Single cell totoRNAseq protocol last used on 06142013 (and 06142013b) - try to decrease primer dimers | *Optimize Single cell totoRNAseq protocol last used on 06142013 (and 06142013b) | ||
*Reduce | *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): == | == Library Preparation (totoRNAseq): == | ||
Line 102: | Line 98: | ||
::{| {{table}} | ::{| {{table}} | ||
|- | |- | ||
|||||||||||<u>Odd Wells</u>|<u>Even Wells</u> | |||||||||||<u>Odd Wells</u>||||||||||<u>Even Wells</u> | ||
|- | |- | ||
|fRNA||||||||||2ul||2ul | |fRNA||||||||||2ul||||||||||2ul | ||
|- | |- | ||
|1mM ATP||||||||||0.5ul||0. | |1mM ATP||||||||||0.5ul||||||||||0.4ul | ||
|- | |- | ||
|100mM DTT||||||||||0.15ul||0. | |100mM DTT||||||||||0.15ul||||||||||0.1ul | ||
|- | |- | ||
|Water||||||||||1.35ul||0ul | |Water||||||||||1.35ul||||||||||0ul | ||
|- | |- | ||
|RNase Inhibitor(1:10 or 4U/ul)||||||||||0.5ul||0.1ul ( | |RNase Inhibitor(1:10 or 4U/ul)||||||||||0.5ul||||||||||0.1ul (1:2 dil) | ||
|- | |- | ||
|PNK||||||||||0.5ul||0.4ul | |PNK||||||||||0.5ul||||||||||0.4ul | ||
|- | |- | ||
|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> | |||
|- | |||
|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''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Odd Wells</u>||||||||||<u>Even Wells</u> | |||
|- | |||
|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'' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Odd Wells</u>||||||||||<u>Even Wells</u> | |||
|- | |||
|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)''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|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)''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|TSO Reaction||||||||||11ul | |||
|- | |||
|2x Buffer||||||||||25ul | |||
|- | |||
|10uM PB_PCR_Fmod||||||||||1ul | |||
|- | |||
|10uM PCR_N2_idX||||||||||1ul | |||
|- | |||
|H2O||||||||||12ul | |||
|- | |||
|Total||||||||||50ul | |||
|- | |||
|} | |||
::{| {{table}} | |||
|- | |||
|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: Will send these libraries for sequencing''' | |||
[[File:07252013_PCR2_gel1.jpg]][[File:07252013_PCR2.jpg]] |
Latest revision as of 00:15, 30 July 2013
RNA-Seq Library Information[edit]
Library ID | Samples | Input RNA | TSO Primer | Bar Code Primers | Bar Code Primer 2 | Type of Seq | Reads | Library Conc |
BL_mNPC_SingleCell_0725 | mNPC | Single cell | TSO.r04 | T20V.id1-96 | N2.id81-82 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read | |
mNPC | 5-10 cells | |||||||
UHRR | 10pg | |||||||
Plate Layout: Samples and Indexes[edit]
' | 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[edit]
- 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):[edit]
- 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[edit]
- 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:
Note: Will send these libraries for sequencing