Blue:RNA-Seq Experiments:08202013: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''Library Conc''' | | align="center" style="background:#f0f0f0;"|'''Library Conc''' | ||
|- | |- | ||
| | | BL_huES8_SingleCell_N2id85_0820||huES8||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.id1-24]||[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|| | ||
|- | |- | ||
| || | | BL_huES8_SingleCell_N2id86_0820||huES8||Single Cell||TSO.R04||T20V.id25-48||N2.id86|||||| | ||
|- | |- | ||
| || | | BL_huES8_SingleCell_N2id87_0820||UHRR||250pg||TSO.R04||T20V.id1||N2.id87|||||| | ||
|- | |- | ||
| ||UHRR|| | | BL_huES8_SingleCell_N2id88_0820||UHRR||250pg||TSO.R04||T20V.id2||N2.id88|||||| | ||
|- | |||
| ||huES8||Single cell||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#IVT_5.27_primer_sequence RL.IVT.N9]||T20V.id49-72||N2.id89|||||| | |||
|- | |||
| ||huES8||Single cell||RL.IVT.N9||T20V.id73-96||N2.id90|||||| | |||
|- | |||
| ||UHRR||250pg||RL.IVT.N9||T20V.id1||N2.id91|||||| | |||
|- | |||
| ||UHRR||250pg||RL.IVT.N9||T20V.id2||N2.id92|||||| | |||
|- | |- | ||
|} | |} | ||
Line 67: | Line 74: | ||
== Purpose == | == Purpose == | ||
*Repeat Single cell totoRNAseq protocol last used on 06142013b | *Repeat Single cell RNAseIII/TSO totoRNAseq protocol last used on 06142013b and compared to 250pg UHRR | ||
**Test Decrease in T20 concentration 0.1uM | **Test Decrease in T20 concentration 0.1uM | ||
**Test Decrease in total reaction volume for RT reaction | **Test Decrease in total reaction volume for RT reaction | ||
*Test | *Test single cell RNaseIII/IVT protocol compated to 250pg of UHRR | ||
*Sort into minimal volume of lysis buffer (0.4ul - bubbled) to keep downstream reaction volumes low | |||
== Library Preparation (totoRNAseq): == | == Library Preparation (totoRNAseq): == | ||
Line 101: | Line 109: | ||
|RNA||||||||||0.4ul | |RNA||||||||||0.4ul | ||
|- | |- | ||
|10x RNaseIII Buffer||||||||||0. | |10x RNaseIII Buffer||||||||||0.2ul | ||
|- | |- | ||
|0.1uM T20V.id||||||||||0.1ul | |0.1uM T20V.id||||||||||0.1ul | ||
|- | |- | ||
|dH20||||||||||0. | |dH20||||||||||0.55ul | ||
|- | |- | ||
|Total||||||||||1.25ul | |Total||||||||||1.25ul | ||
Line 196: | Line 204: | ||
|Betaine (5M)||||||||||2ul | |Betaine (5M)||||||||||2ul | ||
|- | |- | ||
|RNase Inhibitor (40U/ul)||||||||||0. | |RNase Inhibitor (40U/ul)||||||||||0.1ul | ||
|- | |- | ||
|Smarter MMLV (200U/ul)||||||||||0. | |Smarter MMLV (200U/ul)||||||||||0.23ul | ||
|- | |- | ||
|Total||||||||||10ul | |Total||||||||||10ul | ||
Line 216: | Line 224: | ||
::#Air Dry 10min or until cracking first occurs | ::#Air Dry 10min or until cracking first occurs | ||
::#Resuspend beads in 20ul dH2O, incubate 2min, transfer to new tube | ::#Resuspend beads in 20ul dH2O, incubate 2min, transfer to new tube | ||
::#Vacuspin room temperature for 10+min to bring volume to 6ul (TSO) or 7.7ul (IVT) | |||
== TSO == | == TSO == | ||
Line 271: | Line 277: | ||
|2x Buffer||||||||||25ul | |2x Buffer||||||||||25ul | ||
|- | |- | ||
|10uM | |10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence PB_PCR_F]||||||||||1ul | ||
|- | |- | ||
|10uM PCR_N2_idX||||||||||1ul | |10uM PCR_N2_idX||||||||||1ul | ||
Line 301: | Line 307: | ||
:::*Resuspend in 10ul | :::*Resuspend in 10ul | ||
:::*Run 1ul on gel | :::*Run 1ul on gel | ||
[[File:08202013_TSO_PCR1_gel1.jpg]] | |||
== IVT: == | == IVT: == | ||
:'''2nd Strand''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|Purified cDNA||||||||||7.7ul | |||
|- | |||
|FS Buffer||||||||||0.2ul | |||
|- | |||
|SS Buffer||||||||||1ul | |||
|- | |||
|dNTP||||||||||0.8ul | |||
|- | |||
|DNA Pol||||||||||0.2ul | |||
|- | |||
|RNase H||||||||||0.1ul | |||
|- | |||
|Total||||||||||10ul | |||
|- | |||
|} | |||
:::''Incubate @16C 2hours'' | |||
:'''Add T7 Promoter''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|dsDNA||||||||||10ul | |||
|- | |||
|KAPA qPCR MM||||||||||25ul | |||
|- | |||
|[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#IVT_3.27_primer_sequence RL.Add.T7] (10uM)||||||||||0.5ul | |||
|- | |||
|dH2O||||||||||14.5ul | |||
|- | |||
|Total||||||||||50ul | |||
|- | |||
|} | |||
:::''Incubate @94C 2min'' | |||
:::''Incubate @60C 2min'' | |||
:::''Incubate @72C 5min'' | |||
:'''Beads Purify''' | |||
::*Add 50ul beads | |||
::*Wash twice with 80% EtOH | |||
::*Resuspend in 6.4ul | |||
:'''IVT RXN''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|dsDNA||||||||||6.4ul | |||
|- | |||
|ATP||||||||||1.6ul | |||
|- | |||
|GTP||||||||||1.6ul | |||
|- | |||
|CTP||||||||||1.6ul | |||
|- | |||
|UTP||||||||||1.6ul | |||
|- | |||
|10x T7 Buffer||||||||||1.6ul | |||
|- | |||
|T7 Polymerase||||||||||1.6ul | |||
|- | |||
|Total||||||||||16ul | |||
|- | |||
|} | |||
:::''Incubate @37C 10 hours; lid 70C'' | |||
:::''Incubate @4C until ready'' | |||
:'''RT RXN''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|synthesized RNA||||||||||5ul | |||
|- | |||
|10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#IVT_5.27_primer_sequence RL.IVT.N9]||||||||||1ul | |||
|- | |||
|Water||||||||||1ul | |||
|- | |||
|} | |||
:::''Incubate @65C 5min'' | |||
:::''Incubate Ice 1min'' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|10x RT Buffer||||||||||1ul | |||
|- | |||
|10mM dNTP||||||||||0.5ul | |||
|- | |||
|100mM DTT||||||||||0.5ul | |||
|- | |||
|RNase Inh||||||||||0.5ul | |||
|- | |||
|SSIII||||||||||0.5ul | |||
|- | |||
|Total||||||||||10ul | |||
|- | |||
|} | |||
:::''Incubate @25C 5min'' | |||
:::''Incubate @50C 20min'' | |||
:::''Incubate @70C 10min'' | |||
:'''Beads Purify''' | |||
::*Add 10ul beads | |||
::*Wash twice with 80% EtOH | |||
::*Resuspend in 23ul dH2O | |||
:'''PCR (PhusionHF) and SYBR''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|SSIII Reaction||||||||||23ul | |||
|- | |||
|2x Buffer||||||||||25ul | |||
|- | |||
|10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence PB_PCR_F]||||||||||1ul | |||
|- | |||
|10uM PCR_N2_idX||||||||||1ul | |||
|- | |||
|Total||||||||||50ul | |||
|- | |||
|} | |||
::{| {{table}} | |||
|- | |||
|98C 30 sec|||||||||| | |||
|- | |||
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x | |||
|- | |||
|98C 10s -> 72C 20s||||||||||10x (9x for Sybr) | |||
|- | |||
|72C, 5min | |||
|- | |||
|} | |||
:::*Ampure Beads Purify Phusion Amplified(0.8x) | |||
:::*Resuspend in 10ul | |||
:::*Run 1ul on gel | |||
[[File:08202013_IVT_PCR1_gel1.jpg]] | |||
== Results of Amplification == | == Results of Amplification == | ||
*Ran 1ul on gel: | *Ran 1ul on gel: | ||
See "Final Library Preparation" below | |||
== Final Library Preparation == | == Final Library Preparation == | ||
Line 319: | Line 509: | ||
|2xSybr Buffer||||||||||25ul | |2xSybr Buffer||||||||||25ul | ||
|- | |- | ||
|10uM | |10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence ILMN_PCR_F]||||||||||1ul | ||
|- | |- | ||
|10uM | |10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence ILMN_PCR_R]||||||||||1ul | ||
|- | |- | ||
|dH2O||||||||||22ul | |dH2O||||||||||22ul | ||
Line 338: | Line 528: | ||
*Resuspended in 10ul and ran 1 ul on gel: | *Resuspended in 10ul and ran 1 ul on gel: | ||
[[File:08202013_PCR2_gel1.jpg]] | |||
*Re-Amplified 1ul of original id85 and id86 libraries (beads purified 0.8x) and combined with re-amplified libraries shown above | |||
*Size selection to remove primer dimer band: | |||
[[File:08202013_size_sel_gel1.jpg]] [[File:08202013_size_sel_gel2.jpg]] |
Latest revision as of 21:33, 3 September 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_huES8_SingleCell_N2id85_0820 | huES8 | Single cell | TSO.r04 | T20V.id1-24 | N2.id85 | totoRNAseq | Read1 plus N2 barcode read and Read2 for T20 barcode read | |
BL_huES8_SingleCell_N2id86_0820 | huES8 | Single Cell | TSO.R04 | T20V.id25-48 | N2.id86 | |||
BL_huES8_SingleCell_N2id87_0820 | UHRR | 250pg | TSO.R04 | T20V.id1 | N2.id87 | |||
BL_huES8_SingleCell_N2id88_0820 | UHRR | 250pg | TSO.R04 | T20V.id2 | N2.id88 | |||
huES8 | Single cell | RL.IVT.N9 | T20V.id49-72 | N2.id89 | ||||
huES8 | Single cell | RL.IVT.N9 | T20V.id73-96 | N2.id90 | ||||
UHRR | 250pg | RL.IVT.N9 | T20V.id1 | N2.id91 | ||||
UHRR | 250pg | RL.IVT.N9 | T20V.id2 | N2.id92 |
Plate Layout: Samples and Indexes[edit]
' | 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[edit]
- Repeat Single cell RNAseIII/TSO totoRNAseq protocol last used on 06142013b and compared to 250pg UHRR
- Test Decrease in T20 concentration 0.1uM
- Test Decrease in total reaction volume for RT reaction
- Test single cell RNaseIII/IVT protocol compated to 250pg of UHRR
- Sort into minimal volume of lysis buffer (0.4ul - bubbled) to keep downstream reaction volumes low
Library Preparation (totoRNAseq):[edit]
- 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 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) 0.1ul Smarter MMLV (200U/ul) 0.23ul Total 10ul
- Incubate @ 42C 30 min
- Beads Purification
- Add 15ul beads per well
- Allow to bind 15 min
- 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) or 7.7ul (IVT)
TSO[edit]
- 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_F 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
File:08202013 TSO PCR1 gel1.jpg
IVT:[edit]
- 2nd Strand
Volume Purified cDNA 7.7ul FS Buffer 0.2ul SS Buffer 1ul dNTP 0.8ul DNA Pol 0.2ul RNase H 0.1ul Total 10ul
- Incubate @16C 2hours
- Add T7 Promoter
Volume dsDNA 10ul KAPA qPCR MM 25ul RL.Add.T7 (10uM) 0.5ul dH2O 14.5ul Total 50ul
- Incubate @94C 2min
- Incubate @60C 2min
- Incubate @72C 5min
- Beads Purify
- Add 50ul beads
- Wash twice with 80% EtOH
- Resuspend in 6.4ul
- IVT RXN
Volume dsDNA 6.4ul ATP 1.6ul GTP 1.6ul CTP 1.6ul UTP 1.6ul 10x T7 Buffer 1.6ul T7 Polymerase 1.6ul Total 16ul
- Incubate @37C 10 hours; lid 70C
- Incubate @4C until ready
- RT RXN
Volume synthesized RNA 5ul 10uM RL.IVT.N9 1ul Water 1ul
- Incubate @65C 5min
- Incubate Ice 1min
Volume 10x RT Buffer 1ul 10mM dNTP 0.5ul 100mM DTT 0.5ul RNase Inh 0.5ul SSIII 0.5ul Total 10ul
- Incubate @25C 5min
- Incubate @50C 20min
- Incubate @70C 10min
- Beads Purify
- Add 10ul beads
- Wash twice with 80% EtOH
- Resuspend in 23ul dH2O
- PCR (PhusionHF) and SYBR
Volume SSIII Reaction 23ul 2x Buffer 25ul 10uM PB_PCR_F 1ul 10uM PCR_N2_idX 1ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 10x (9x for Sybr) 72C, 5min
- Ampure Beads Purify Phusion Amplified(0.8x)
- Resuspend in 10ul
- Run 1ul on gel
File:08202013 IVT PCR1 gel1.jpg
Results of Amplification[edit]
- Ran 1ul on gel:
See "Final Library Preparation" below
Final Library Preparation[edit]
- Re-Amplified 1ul using Sybr-Green and ILMN_PCR_F/R
Volume Library 1ul 2xSybr Buffer 25ul 10uM ILMN_PCR_F 1ul 10uM ILMN_PCR_R 1ul dH2O 22ul Total 50ul
- 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 1ul of original id85 and id86 libraries (beads purified 0.8x) and combined with re-amplified libraries shown above
- Size selection to remove primer dimer band:
File:08202013 size sel gel1.jpg File:08202013 size sel gel2.jpg