Editing
Blue:RNA-Seq Experiments:08202013
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
== RNA-Seq Library Information == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Input RNA''' | align="center" style="background:#f0f0f0;"|'''TSO Primer''' | align="center" style="background:#f0f0f0;"|'''Bar Code Primers''' | align="center" style="background:#f0f0f0;"|'''Bar Code Primer 2''' | align="center" style="background:#f0f0f0;"|'''Type of Seq''' | align="center" style="background:#f0f0f0;"|'''Reads''' | 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|||||| |- | 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|||||| |- |} == 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 Cells Sorted [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:08122013 08122013] == Purpose == *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): == :'''Cell Sort''' ::{| {{table}} |- |||||||||||<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 |- |0.1uM T20V.id||||||||||0.1ul |- |dH20||||||||||0.55ul |- |Total||||||||||1.25ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1min'' :::''Incubate @ 4C 10min'' :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA||||||||||4.1ul |- |10mM dNTP Mix||||||||||1ul |- |dH2O||||||||||0ul |- |0.1uM T20V.ID||||||||||0.9ul |- |} :::''Incubate @ 65C 3 min'' :::''Transfer to Ice'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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 == :'''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 [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 |- |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 [[File:08202013_TSO_PCR1_gel1.jpg]] == 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 == *Ran 1ul on gel: See "Final Library Preparation" below == Final Library Preparation == *Re-Amplified 1ul using Sybr-Green and ILMN_PCR_F/R ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Library||||||||||1ul |- |2xSybr Buffer||||||||||25ul |- |10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence ILMN_PCR_F]||||||||||1ul |- |10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence 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: [[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]]
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Template used on this page:
Template:Table
(
edit
)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information