Editing
Blue:RNA-Seq Experiments:06042014
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!
== Purpose == *Improve upon totoQuartzSeq protocol - optimize PCR Cylcles for 1ng UHRR == RNA-Seq Library Information == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Condition''' | align="center" style="background:#f0f0f0;"|'''5' Adaptor''' | align="center" style="background:#f0f0f0;"|'''3' Adaptor''' | align="center" style="background:#f0f0f0;"|'''N2 PCR Rev Primer''' | align="center" style="background:#f0f0f0;"|'''RNA-Seq Protocol''' |- | ||2x1ng UHRR||RIII Frag - 11 PCR cycles||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||2x1ng UHRR||RIII Frag - 13 PCR cycles||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||2x1ng UHRR||RIII Frag - 15 PCR cycles||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||NTC||RIII Frag - 15 PCR cycles||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||2x1ng UHRR||Mg Frag - 11 PCR cycles||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||2x1ng UHRR||Mg Frag - 13 PCR cycles||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||2x1ng UHRR||Mg Frag - 15 PCR cycles||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- | ||NTC||Mg Frag - 15 PCR cycles||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq |- |} Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence] == cDNA Preparation: RNAseIII-Based Fragmentation == :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2ng/ul UHRR ||||||||||0.5ul |- |1:10e4 ERCC ||||||||||0.2ul |- |0.1uM Quartz-RT (see below)||||||||||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 1 min'' :'''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 4-10 min'' :::''Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]'' :'''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 60 min'' :'''Beads Purification''' ::#Add 25ul 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 ExoI reaction (see below): == cDNA Preparation: Mg-based Fragmentation == :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2ng/ul UHRR ||||||||||0.5ul |- |1:10e4 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 |- |40U/ul 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 |- |600U/ul Affymetrix PAP||||||||||0.2ul |- |dH2O||||||||||0.2ul |- |Total|||||||||| 4 |- |} :::''Incubate @ 37C 10 min'' :::''Transfer to Ice'' :::''Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||5ul |- |5xRT Buffer||||||||||1ul |- |10mM dNTP Mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |Betaine (5M)||||||||||2ul |- |40U/ul Rnase Inhibitor||||||||||0.1ul |- |Smarter MMLV (200U/ul)||||||||||0.25ul |- |dH2O||||||||||0.4ul |- |Total||||||||||10ul |- |} :::''Incubate @ 42C 60 min'' :'''Beads Purification''' ::#Add 25ul 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 ExoI reaction (below): == Quartz-Seq Library Preparation == '''RT Primer Removal''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x PCR Buffer (homemade Takara)||||||||||0.4ul |- |10x Exo I buffer (epicentre)||||||||||0.2ul |- |100mM DTT||||||||||0.2ul |- |Exonuclease I (epicentre)||||||||||0.6ul |- |dH2O||||||||||4.6ul |- |Total||||||||||6ul |- |} ::::''Takara Taq PCR Buffer:'' ::::{| {{table}} |- |||||||||||<u>Volume</u> |- |1M Tris.HCl (pH 8.3)||||||||||100ul (100mM Final) |- |1M KCl||||||||||500ul (500mM Final) |- |1M MgCl2||||||||||15ul (15mM Final) |- |dH2O||||||||||385ul |- |Total||||||||||1ml |- |} ::: Add directly to beads and mix :::''Incubate @37C 30min'' :::''Incubate @80C 20min'' :::''Incubate @4C'' '''Poly-A Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |ExoI RXN||||||||||6ul |- |10x PCR Buffer||||||||||0.5ul |- |10mM dATP||||||||||1.5ul |- |RNaseH (ambion)||||||||||0.12ul |- |TdT enzyme (Roche 03333566001)||||||||||0.42ul |- |dH2O||||||||||2.46ul |- |Total||||||||||11ul |- |} :::''Incubate @37C 50 sec'' :::''Incubate @65C 10min'' :::''Incubate @4C'' '''Second Strand Synthesis''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Poly-A Tailed Sample||||||||||11ul |- |2x Terra Direct PCR Buffer||||||||||25ul |- |10uM Tagging Primer||||||||||0.32ul |- |Terra Polymerase||||||||||2ul |- |dH2O||||||||||18.68ul |- |Total||||||||||57ul |- |} :::''Incubate @98C 2min 10 sec'' :::''Incubate @40C 1min'' :::''Incubate @68C 5min'' :::''Transfer to ice'' '''PCR Amplification''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2nd Strand Sample||||||||||57ul |- |2x Terra Direct PCR Buffer||||||||||25ul |- |100uM PCR Primer||||||||||1ul |- |dH2O||||||||||24ul |- |Total||||||||||107ul |- |} ::{| {{table}} |- |68C 1 sec|||||||||| |- |98C 10s -> 65C 15s -> 68C 5min||||||||||12-15x (1ng) |- |68C, 5min |- |} == Library Quantification and Nextera Preparation == *Beads purify once (0.8x) and reconstitute in 10ul *Run 1ul on gel: [[File:06052014_PCR1_Gel1.jpg|500px]] *Quantify 1ul using Qubit: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Condition''' | align="center" style="background:#f0f0f0;"|'''[cDNA] ng/ul''' | align="center" style="background:#f0f0f0;"|'''Volume for 0.2ng/ul''' | align="center" style="background:#f0f0f0;"|'''Volume dH2O (final 5ul)''' | align="center" style="background:#f0f0f0;"|'''Nextera Barcode''' |- | 1|| RIII (11)||0.726||1.38||3.62||N503/N701 |- | 2|| RIII (11)||0.541||1.85||3.15||N503/N702 |- | 3|| RIII (13)||1.14||0.88||4.12||N503/N703 |- | 4|| RIII (13)||0.938||1.07||3.93||N503/N704 |- | 5|| RIII (15)||2.13||0.47||4.53||N503/N705 |- | 6|| RIII (15)||2.59||0.39||4.61||N503/N706 |- | 7|| RIII NTC (15)||0.41||2.44||2.56||N503/N707 |- | 8|| RIII NTC (15)||0.508||1.97||3.03||N503/N708 |- | 9|| Mg (11)||0.366||-||-|| |- | 10|| Mg (11)||0.559||-||-|| |- | 11|| Mg (13)||0.52||-||-|| |- | 12|| Mg (13)||0.45||-||-|| |- | 13|| Mg (15)||1.11||0.90||4.10||N504/N709 |- | 14|| Mg (15)||0.9||1.11||3.89||N504/N710 |- | 15|| Mg NTC (15)||0.312||3.21||1.79||N504/N711 |- | 16|| Mg NTC (15)||0.457||2.19||2.81||N504/N712 |- | |} ''Nextera Tagmentation'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Tagment DNA Buffer||||||||||5ul |- |Amplification Tagment Mix||||||||||2.5ul |- |Diluted Sample||||||||||2.5ul |- |Total||||||||||10ul |- |} ::*Vortex and spin ::*Incubate 55C 5 min ::*Hold 10C ::*Once sample reaches 10C add 2.5ul of NT buffer in block ::*Vortex and spin ''Nextera Amplification'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Tagmented Sample||||||||||12.5ul |- |Nextera PCR Mix||||||||||7.5ul |- |S5xx Primer||||||||||2.5ul |- |S7xx Primer||||||||||2.5ul |- |50x Sybr green||||||||||0.08ul |- |Total||||||||||25ul |- |} ::''Mix and centrifuge'' ::{| {{table}} |- |72C 3 min|||||||||| |- |95C 30s|||||||||| |- |95C 10s -> 55C 30s -> 72C 60s||||||||||8x |- |72C, 5min |- |} ::*Run 2.5ul on gel: [[File:06042014_PCR2_Gel1.jpg|500px]] :*Results: Can see that the reduction of the nextera cycles resulted in a lower cDNA yield compared to 03052014 using 12 cycles - hopefully this will result in fewer clonal arrays in the final libraries :*Combined 5ul each (10ul for 9 and 10) :*beads purified twice (0.8x volume) :*Resuspended in 10ul, ran 1ul on gel: [[File:06052014_PCR2_Gel1.jpg|75px]]
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