Blue:RNA-Seq Experiments:10282013: Difference between revisions

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>B1lake
(Created page with " == RNA-Seq Library Information == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Samples''' | al...")
 
>B1lake
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| align="center" style="background:#f0f0f0;"|'''Library Conc'''
| align="center" style="background:#f0f0f0;"|'''Library Conc'''
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| BL_||UHRR/ERCC||100pg - RNase3 2 min||TSO.r06||T20V.id2||N2.id1-2||totoRNAseq||||
| BL_||UHRR/ERCC||100pg - RNase3 2 min/PAP 5min||TSO.r06||T20V.id1-8||N2.id1||totoRNAseq||||
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| BL_||UHRR/ERCC||100pg - RNase3 4 min||TSO.r06||T20V.id2||N2.id3-4||totoRNAseq||||
| BL_||UHRR/ERCC||100pg - RNase3 2 min/PAP 10min||TSO.r06||T20V.id9-16||N2.id2||totoRNAseq||||
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| BL_||UHRR/ERCC||100pg - RNase3 6 min||TSO.r06||T20V.id2||N2.id5-6||totoRNAseq||||
| BL_||UHRR/ERCC||100pg - RNase3 2 min/PAP 30min||TSO.r06||T20V.id17-24||N2.id3||totoRNAseq||||
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| BL_||UHRR/ERCC||100pg - RNase3 6 min - 0.01/1 3'-dATP/ATP||TSO.r06||T20V.id2||N2.id7-8||totoRNAseq||||
| BL_||UHRR/ERCC||100pg - RNase3 5 min/PAP 5min||TSO.r06||T20V.id25-32||N2.id4||totoRNAseq||||
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| BL_||UHRR/ERCC||100pg - RNase3 6 min - 0.02/1 3'-dATP/ATP||TSO.r06||T20V.id2||N2.id9-10||totoRNAseq||||
| BL_||UHRR/ERCC||100pg - RNase3 5 min/PAP 10min||TSO.r06||T20V.id33-40||N2.id5||totoRNAseq||||
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| BL_||UHRR/ERCC||100pg - RNase3 6 min - 0.05/1 3'-dATP/ATP||TSO.r06||T20V.id2||N2.id11-12||totoRNAseq||||
| BL_||UHRR/ERCC||100pg - RNase3 5 min/PAP 30min||TSO.r06||T20V.id41-48||N2.id6||totoRNAseq||||
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| BL_||UHRR/ERCC||100pg - RNase3 6 min - 0.1/1 3'-dATP/ATP||TSO.r06||T20V.id2||N2.id13-14||totoRNAseq||||
| BL_||UHRR/ERCC||100pg - RNase3 10 min/PAP 5min||TSO.r06||T20V.id49-56||N2.id7||totoRNAseq||||
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| BL_||UHRR/ERCC||100pg - RNase3 6 min - 0.5/1 3'-dATP/ATP||TSO.r06||T20V.id2||N2.id15-16||totoRNAseq||||
| BL_||UHRR/ERCC||100pg - RNase3 10 min/PAP 10min||TSO.r06||T20V.id57-64||N2.id8||totoRNAseq||||
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| BL_||UHRR/ERCC||100pg - RNase3 6 min - Affymetrix PAP||TSO.r06||T20V.id2||N2.id17-18||totoRNAseq||||
| BL_||UHRR/ERCC||100pg - RNase3 10 min/PAP 30min||TSO.r06||T20V.id64-72||N2.id9||totoRNAseq||||
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| BL_||UHRR/ERCC||100pg - RNase3 6 min - Affymetrix PAP - heat 65C 10min||TSO.r06||T20V.id2||N2.id19-20||totoRNAseq||||
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|}
|}
Note: Each condition includes 8 ATP variables:
#1mM ATP/0mM 3'dATP
#1mM ATP/0.01mM 3'dATP
#1mM ATP/0.05mM 3'dATP
#1mM ATP/0.1mM 3'dATP
#0.1mM ATP/0mM 3'dATP
#0.1mM ATP/0.001mM 3'dATP
#0.1mM ATP/0.005mM 3'dATP
#0.1mM ATP/0.01mM 3'dATP


== Purpose ==
== Purpose ==

Revision as of 23:19, 25 October 2013

RNA-Seq Library Information

Library ID Samples Input RNA TSO Primer Bar Code Primers Bar Code Primer 2 Type of Seq Reads Library Conc
BL_ UHRR/ERCC 100pg - RNase3 2 min/PAP 5min TSO.r06 T20V.id1-8 N2.id1 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 2 min/PAP 10min TSO.r06 T20V.id9-16 N2.id2 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 2 min/PAP 30min TSO.r06 T20V.id17-24 N2.id3 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 5 min/PAP 5min TSO.r06 T20V.id25-32 N2.id4 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 5 min/PAP 10min TSO.r06 T20V.id33-40 N2.id5 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 5 min/PAP 30min TSO.r06 T20V.id41-48 N2.id6 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 10 min/PAP 5min TSO.r06 T20V.id49-56 N2.id7 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 10 min/PAP 10min TSO.r06 T20V.id57-64 N2.id8 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3 10 min/PAP 30min TSO.r06 T20V.id64-72 N2.id9 totoRNAseq


Note: Each condition includes 8 ATP variables:

  1. 1mM ATP/0mM 3'dATP
  2. 1mM ATP/0.01mM 3'dATP
  3. 1mM ATP/0.05mM 3'dATP
  4. 1mM ATP/0.1mM 3'dATP
  5. 0.1mM ATP/0mM 3'dATP
  6. 0.1mM ATP/0.001mM 3'dATP
  7. 0.1mM ATP/0.005mM 3'dATP
  8. 0.1mM ATP/0.01mM 3'dATP

Purpose

  • To try and reduce the number of PolyA reads in the totoRNAseq library that might arise from overfragmentation or overactive PAP
    • Test RNaseIII fragmentation times - want time that doesn't overfragment but gives sharper distribution in sizes around 100-200 bases
    • Test reducing ATP to more limiting 0.1mM
    • Test termination of PAP activity to limit poly-A tail length by adding Cordycepin-ATP or 3'-ATP at different ratios to regular ATP
    • Test PAP incubation times - does longer PAP treatment lead to an increase in lower copy transcripts?
    • Include PAP inactivation - heating


RNAseIII-Fragmented Library Preparation

RNA
Volume
UHRR 200pg/ul 0.5ul
ERCC 1:10E5 0.2ul
10x RNaseIII Buffer 0.2ul
0.1uM T20V.id 0.1ul
dH20 0.25ul
Total 1.25ul
Incubate @ 72C 3 min
Incubate @ 37C 1min


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
fRNA 3.1ul
5x RT Buffer 0.5ul
1mM ATP (or mix) 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
0.1uM T20V.ID 0.9ul


Incubate @ 65C 3 min (or 10 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
  1. Add 15ul beads per well
  2. Allow to bind 15 min
  3. Remove supernatant and wash 2x with 1ml 80% EtOH
  4. Air Dry 10min or until cracking first occurs
  5. Resuspend beads in 10ul dH2O, incubate 2min, transfer to new tube - vacuspin 8 min to bring to 3ul


TSO
Volume
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)
Volume
TSO Reaction 11ul
2x Buffer 25ul
10uM P1-STRT 1ul
10uM PCR_R_N2_id 1ul
H2O 12ul
Total 50ul


98C 30 sec
98C 15s -> 58C 20s -> 72C 30s 6x
98C 10s -> 72C 20s 12x
72C, 5min

Results of Amplification

  • Run 5ul on gel:


File:10222013 PCR1 gel1.jpg


Photoshop modified file:


Media:10222013_PCR1_gel1_Mod.jpg

Final Library Preparation

  • Beads purified twice at (0.8x), re-suspend in 10ul