Editing
Blue:RNA-Seq Experiments:01082014
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_totoRNAseq_10pgUHRR_Jan08||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 1min||TSO.r06||T20VN_PE_R||N2.id1-2||totoRNAseq|||| |- | ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 2min||TSO.r06||T20VN_PE_R||N2.id3-4||totoRNAseq|||| |- | ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_PE_R||N2.id5-6||totoRNAseq|||| |- | ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 6min||TSO.r06||T20VN_PE_R||N2.id7-8||totoRNAseq|||| |- | ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 8min||TSO.r06||T20VN_PE_R||N2.id9-10||totoRNAseq|||| |- | ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 10min||TSO.r06||T20VN_PE_R||N2.id11-12||totoRNAseq|||| |- | ||UHRR/ERCC||1ng - RNase3 1min/Affymetrix PAP 10min||TSO.r06||T20VN_PE_R||N2.id13-14||totoRNAseq|||| |- |} Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence] == Purpose == *Test optimal PAP treatment time for 10pg RNA based on [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:12102013 12062013] *Use ERCCs to determine level of transcript amplification between libraries == RNAseIII-Fragmented Library Preparation == :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |UHRR 2ng/ul or 0.02ng/ul ||||||||||0.5ul |- |ERCC 1:10E4 or 1:10E6||||||||||0.2ul |- |0.1uM T20VN||||||||||0.1ul |- |dH2O||||||||||0.2ul |- |Total||||||||||1ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1 min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x RNaseIII buffer||||||||||0.2ul |- |RNase III||||||||||0.1ul |- |dH20||||||||||0.7ul |- |Total||||||||||2ul |- |} :::''Incubate @ 37C 15s-1min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||2ul |- |5x RT buffer||||||||||0.5ul |- |1mM ATP||||||||||0.5ul |- |600U/ul Affymetrix PAP||||||||||0.2ul |- |40U/ul Rnase Inhibitor||||||||||0.2ul |- |dH2O||||||||||0.6ul |- |Total||||||||||4ul |- |} :::''Incubate @ 37C 1-10 min'' :::''Add 1ul of 0.1uM T20VN'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||5ul |- |5xRT Buffer||||||||||1.5ul |- |10mM dNTP Mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |Betaine (5M)||||||||||2ul |- |Smarter MMLV (200U/ul)||||||||||0.25ul |- |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 to 3ul :'''TSO Reaction''' ::{| {{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'' :'''PCR (PhusionHF)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||10ul |- |2x Buffer||||||||||25ul |- |10uM P1-STRT||||||||||1ul |- |10uM PCR_R_N2_id||||||||||1ul |- |H2O||||||||||13ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 72C 20s||||||||||9x (1ng); 15x (10pg) |- |72C, 5min |- |} == Results of Amplification == *Run 5ul on gel: [[File:01082013_PCR1_Gel1.jpg|400px]] Results: Global amplification levels seem a little low, but otherwise looks fine. Some variability with the 10pg that could be due to the beads purification stage following RT... == Final Library Preparation == *Beads purified twice at (0.8x), re-suspended in 10ul *PCR on 0.5ul each library using ILMN_PCR_F and either ERCC130R227 or ERCC130R964. [[File:01082014_ERCC130_R227.jpg|400px]][[File:01082014_ERCC130_R964b.jpg|400px]] Results: *Still variable amplification levels seen with 10pg. More consistency might be achieved by combining 10pg samples for beads purification prior to TSO/PCR *Can see at least one of the 2min or 4 min showing similar levels as the 1ng (especially for the R964 - i.e.3'end) while longer PAP times tended to show poorer amplification curves... therefore, 4 min PAP may be sufficient for 10pg RNA quantities *Its difficult to fully conclude optimal conditions due to the variability... hopefully this is an artifact of the single 10pg beads purification rather than inconsistency/inefficiency of the totoRNAseq reactions at these minimal RNA concentrations
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