Editing
Blue:RNA-Seq Experiments:09102013
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_UHRR_N2id9_0910||UHRR/ERCC||0.5ng - RNaseIII Fragmented||[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.id96]||N2_id9||totoRNAseq||QC Analysis|| |- | BL_UHRR_N2id10_0910||UHRR/ERCC||0.5ng - Mg Fragmented||TSO.R04||T20V.id96||N2_id10|||||| |- | ||UHRR/ERCC||0.5ng - Unfragmented||TSO.R04||T20V.id96||N/A|||||| |- | BL_UHRR_N2id11_0910||UHRR/ERCC||0.5ng - RNaseIII Fragmented||TSO.R05||T20V.id96||N2_id11|||||| |- | BL_UHRR_N2id12_0910||UHRR/ERCC||0.5ng - Mg Fragmented||TSO.R05||T20V.id96||N2_id12|||||| |- | ||UHRR/ERCC||0.5ng - Unfragmented||TSO.R05||T20V.id96||N/A|||||| |- | BL_UHRR_N2id13_0910||UHRR/ERCC||0.5ng - RNAseIII Fragmented||Smarter TSO||T20V.id96||N2_id13|||||| |- | BL_UHRR_N2id14_0910||UHRR/ERCC||0.5ng - Mg Fragmented||Smarter TSO||T20V.id96||N2_id14|||||| |- | ||UHRR/ERCC||0.5ng - Unfragmented||Smarter TSO||T20V.id96||N/A|||||| |- | ||UHRR/ERCC||0.5ng - RNaseIII Fragmented||TSO.R04||Supp.T20.id1||N/A|||||| |- | ||UHRR/ERCC||0.5ng - Mg Fragmented||TSO.R04||Supp.T20.id1||N/A|||||| |- | ||UHRR/ERCC||0.5ng - Unfragmented||TSO.R04||Supp.T20.id1||N/A|||||| |- |} == Purpose == *Examine efficiency of our TSO primers (r04/r05) compared to Smarter TSO using ERCC assay *Examine efficiency of each TSO between fragmented and unfragmented using ERCC assay *Compare RNaseIII and Mg based fragmentation methods == Library Preparation (RNAseIII Fragmentation): == :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |1ng/ul UHRR||||||||||0.5ul |- |ERCC 1:10E4||||||||||0.2ul |- |10x RNaseIII Buffer||||||||||0.2ul |- |1uM T20V.id1 or Supp.T20.id||||||||||0.1ul |- |dH20||||||||||0.25ul |- |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'' == Library Preparation (Mg-based Fragmentation) == :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |1ng/ul UHRR RNA||||||||||0.5ul |- |1:1E104 ERCC||||||||||0.2ul |- |5x RT buffer||||||||||1ul |- |dH2O||||||||||0.3ul |- |Total||||||||||2ul |- |} :::''Incubate @ 94C 2 min'' :::''Transfer to ice'' :'''End Repair''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||2ul |- |1mM ATP||||||||||0.4ul |- |100mM DTT||||||||||0.1ul |- |RNase Inhibitor||||||||||0.1ul |- |PNK||||||||||0.5ul |- |Total||||||||||3.1ul |- |} :::''Incubate @ 37C 30 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PNK_fRNA||||||||||3.1ul |- |1mM ATP||||||||||0.5ul |- |0.5U/ul PAP (1:10d in H2O)||||||||||0.5ul |- |} :::''Incubate @ 37C 5 min'' :::''Transfer to Ice'' == Reverse Transcription: == ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA or unfrag RNA (diluted to 3.6ul in dH2O)||||||||||4.1ul (3.6ul) |- |10mM dNTP Mix||||||||||1ul |- |1uM T20V.ID or Supp.T20.id||||||||||0.9ul |- |} :::''Incubate @ 65C 3 min'' :::''Transfer to Ice'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5xRT Buffer||||||||||1.5ul (RNaseIII); 1ul + 0.5ul dH2O (Mg); 2ul (unfrag) |- |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 == 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||||||||||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]; P1-STRT (Smarter TSO); or PCR_Blue_Short||||||||||1ul |- |10uM PGR_PCR_R or PCR_Blue_Short||||||||||1ul |- |H2O||||||||||12ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 72C 20s||||||||||11x |- |72C, 5min |- |} == Results of Amplification == *Ran 5ul on gel: [[File:09102013_PCR1_gel1.jpg]] == TSO Quantification == *Comparison of Pre-amp and post-amp plots: ::{| {{table}} | align="center" style="background:#f0f0f0;"|'''PCR - 5' TSO''' | align="center" style="background:#f0f0f0;"|'''PCR - 3' T20''' | align="center" style="background:#f0f0f0;"|'''PCR fragment''' |- | TSO+130-5'||T20+130-5'||ERCC-130-5' |- | TSO+130-mid||T20+130-mid||ERCC-130-mid |- | TSO+130-3'||T20+130-3'||ERCC-130-3' |- | |} TSO=ILMN_PCR_F; T20=ILMN_PCR_R *Amplified 1ul TSO (diluted 5ul to 50ul) and 1ul PCR (total volume 50ul) using Sybr-Green: ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Library||||||||||1ul |- |2xSybr Buffer||||||||||12.5ul |- |10uM F primer||||||||||0.5ul |- |10uM R Primer||||||||||0.5ul |- |dH2O||||||||||10.5ul |- |Total||||||||||25ul |- |} :::''95C 30sec'' :::''95C 10s -> 60C 20s -> 72C 30s'' => 40X :::''72C, 5min'' Loading Format for qPCR: {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''RnaseIII''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Mg''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Unfrag''' | align="center" style="background:#f0f0f0;"|'''''' |- | ||align="center" style="background:#f0f0f0;"|5'||align="center" style="background:#f0f0f0;"|mid||align="center" style="background:#f0f0f0;"|3'||align="center" style="background:#f0f0f0;"|5'||align="center" style="background:#f0f0f0;"|mid||align="center" style="background:#f0f0f0;"|3'||align="center" style="background:#f0f0f0;"|5'||align="center" style="background:#f0f0f0;"|mid||align="center" style="background:#f0f0f0;"|3' |- | align="center" style="background:#f0f0f0;"|TSO||r04||r04||r04||r04||r04||r04||r04||r04||r04 |- | align="center" style="background:#f0f0f0;"|TSO||r05||r05||r05||r05||r05||r05||r05||r05||r05 |- | align="center" style="background:#f0f0f0;"|TSO||Smart||Smart||Smart||Smart||Smart||Smart||Smart||Smart||Smart |- | align="center" style="background:#f0f0f0;"|TSO||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp |- | align="center" style="background:#f0f0f0;"|PCR||r04||r04||r04||r04||r04||r04||r04||r04||r04 |- | align="center" style="background:#f0f0f0;"|PCR||r05||r05||r05||r05||r05||r05||r05||r05||r05 |- | align="center" style="background:#f0f0f0;"|PCR||Smart||Smart||Smart||Smart||Smart||Smart||Smart||Smart||Smart |- | align="center" style="background:#f0f0f0;"|PCR||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp |- | |} [[File:09102013_ERCC-R_PCR-F.jpg]] [[File:09102013_ERCC130_Unfrag.jpg]] [[File:09102013_ERCC130_RIII.jpg]] [[File:09102013_ERCC130_Mg.jpg]] == Final Library Preparation == *Beads Purified PCR product (0.8x) and resuspended in 10ul: *Re-Amplified using PB_PCR_F or P1-STRT and PCR_R_N2_id primers: :#r04 - RnaseIII - id9 :#r04 - Mg - id10 :#r05 - RnaseIII - id11 :#r05 - Mg - id12 :#Smart - RnaseIII - id13 :#Smart - Mg - id14 ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Library||||||||||1ul Smart or 4ul r04/r05 |- |2xSybr Buffer||||||||||25ul |- |10uM F primer||||||||||1ul |- |10uM R Primer||||||||||1ul |- |dH2O||||||||||up to 50ul |- |} :::''95C 30sec'' :::''95C 10s -> 60C 20s -> 72C 30s'' => 5X :::''72C, 5min'' *Beads purified (0.8x), resuspended in 10ul and ran 1ul on gel: [[File:09102013_PCR2_gel1.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