Blue:RNA-Seq Experiments:01082014: 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''' | ali...")
 
>B1lake
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| align="center" style="background:#f0f0f0;"|'''Library Conc'''
| align="center" style="background:#f0f0f0;"|'''Library Conc'''
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| BL_totoRNAseq_10pgUHRR_Jan08||UHRR/ERCC||10pg - RNase3 15s (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id89-90||totoRNAseq||||
| BL_totoRNAseq_10pgUHRR_Jan08||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 1min||TSO.r06||T20VN_id1-2||N2.id1||totoRNAseq||||
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| ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 2min||TSO.r06||T20VN_id3-4||N2.id1||totoRNAseq||||
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| ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_id5-6||N2.id1||totoRNAseq||||
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| ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 6min||TSO.r06||T20VN_id7-8||N2.id1||totoRNAseq||||
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| ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 8min||TSO.r06||T20VN_id9-10||N2.id1||totoRNAseq||||
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| ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 10min||TSO.r06||T20VN_id11-12||N2.id1||totoRNAseq||||
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Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence]
Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence]


== Purpose ==
== Purpose ==

Revision as of 01:14, 8 January 2014

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_totoRNAseq_10pgUHRR_Jan08 UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 1min TSO.r06 T20VN_id1-2 N2.id1 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 2min TSO.r06 T20VN_id3-4 N2.id1 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 4min TSO.r06 T20VN_id5-6 N2.id1 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 6min TSO.r06 T20VN_id7-8 N2.id1 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 8min TSO.r06 T20VN_id9-10 N2.id1 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 10min TSO.r06 T20VN_id11-12 N2.id1 totoRNAseq


Primer sequences [1]

Purpose

  • Test optimal PAP treatment time for 10pg RNA based on 12062013
  • Use ERCCs to determine level of transcript amplification between libraries


RNAseIII-Fragmented Library Preparation

RNA
Volume
UHRR 0.02ng/ul 0.5ul
ERCC 1:10E6 0.2ul
0.1uM T20VN 0.1ul
dH2O 0.2ul
Total 1ul


Incubate @ 72C 3 min
Incubate @ 37C 1 min


Volume
10x RNaseIII buffer 0.2ul
RNase III 0.1ul
dH20 0.7ul
Total 2ul


Incubate @ 37C 15s


3' Tailing
Volume
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 10 min
Add 1ul of 0.1uM T20VN


Reverse Transcription
Volume
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
  1. Add 15ul beads per well
  2. Allow to bind 15 min, combine
  3. Remove supernatant and wash 2x with 1ml 80% EtOH
  4. Air Dry 10min or until cracking first occurs
  5. Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin to 3ul


TSO Reaction
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 10ul
2x Buffer 25ul
10uM P1-STRT 1ul
10uM PCR_R_N2_id 1ul
H2O 13ul
Total 50ul


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


Results of Amplification

  • Run 5ul on gel:



Results:


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.


Results: