Blue:RNA-Seq Experiments:11012013

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Revision as of 01:03, 1 November 2013 by >B1lake (→‎RNA-Seq Library Information)
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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(.1U) 1mM MnCl2 inactivated @ 65C 10 min/PAP 10min ATP TSO.r06 T20V.id2 N2.id81 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 1mM MnCl2 inactivated @ 65C 10 min/PAP 10min ATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.1U) 1mM MnCl2 inactivated @ 65C 10 min/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id81 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 1mM MnCl2 inactivated @ 65C 10 min/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.1U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP TSO.r06 T20V.id2 N2.id81 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.01U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP TSO.r06 T20V.id2 N2.id81 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.1U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id81 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.01U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id81 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.1U) 5mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 5mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.1U) 5mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 5mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.1U) 10mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 10mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(.1U) 10mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id82 totoRNAseq
BL_ UHRR/ERCC 100pg - RNase3(0U) 10mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP TSO.r06 T20V.id2 N2.id82 totoRNAseq


Note: ATP Condtions:

  1. 1mM ATP/0mM 3'dATP
  2. 1mM ATP/0.05mM 3'dATP

Note: for 5mM and 10mM - no further MnCl2 added to the PAP reaction

Purpose

  • Continuation of tests from 10-30-2013
  • To try and reduce the number of PolyA reads in the totoRNAseq library that might arise from overfragmentation or overactive PAP
    • Reduce chemically induced fragmentation by replacing 65C incubation with RNase Inhibitor
    • Test termination of PAP activity to limit poly-A tail length by adding Cordycepin-ATP or 3'-ATP - can the ~200bp band get reduced by decreasing amount of fragmentation?
    • Exclude PAP inactivation - reduced pre-RT heating (only 65C for 3 minutes)


RNAseIII-Fragmented Library Preparation

RNA
Volume
UHRR 200pg/ul 0.5ul
ERCC 1:10E5 0.2ul
0.1uM T20V.id2 0.1ul
5x Ambion PAP buffer 0.08ul
100mM DTT 0.02ul
dH20 0.27ul
Total 1.17ul
Incubate @ 72C 3 min
Incubate @ 37C 1min


RNA FRAGMENTATION
Volume
RNA 1.17ul
25mM MnCl2 0.08ul or 0.16ul
or 25mM MgCl2 0.16ul
RNase III 0.1ul or 0.01ul
dH20 0.67ul or up to 0.83ul
Total 2ul


Incubate @ 37C 5-10 min


3' Tailing
Volume
fRNA 2ul
5x Ambion PAP Buffer 0.72ul (or 0.8ul)
25mM MnCl2 0.32ul (or 0.4ul)
ATP (or mix) 0.25ul
2U/ul PAP 0.2ul
dH2O or 40U/ul Rnase Inhibitor 0.2ul
dH2O 0.31ul (or 0.15ul)


Incubate @ 37C 10 min
Transfer to Ice


Reverse Transcription
Volume
PAP RNA 4ul
10mM dNTP Mix 1ul
0.2uM T20V.ID 0.5ul


Incubate @ 65C 3 min
Transfer to Ice


Volume
5xRT Buffer 2ul
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 - combine sets
  3. Remove supernatant and wash 2x with 1ml 80% EtOH
  4. Air Dry 10min or until cracking first occurs
  5. Resuspend beads in 3ul dH2O, incubate 2min, transfer to new tube


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 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 12x
72C, 5min

Results of Amplification

  • Run 5ul on gel:


Final Library Preparation

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