Blue:RNA-Seq Experiments:10292013: Difference between revisions
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[[File:10292013_PCR1_gel1.jpg]] | [[File:10292013_PCR1_gel1.jpg]] | ||
Note: Numbers refer to: | |||
#id10 RNaseIII (0.08U)/1mM ATM | |||
#id11 RNaseIII (0.08U)/1mM:0.01mM ATM:3'dATP | |||
#id12 RNaseIII (0.08U)/1mM:0.05mM ATM:3'dATP | |||
#id13 RNaseIII (0.08U)/1mM:0.1mM ATM:3'dATP | |||
#id14 RNaseIII (0.04U)/1mM:0.05mM ATM:3'dATP | |||
#id15 RNaseIII (0.01U)/1mM:0.05mM ATM:3'dATP | |||
#id16 No RNaseIII/1mM:0.05mM ATM:3'dATP | |||
#id17 NC | |||
== Final Library Preparation == | == Final Library Preparation == | ||
*Beads purified twice at (0.8x), re-suspend in 10ul | *Beads purified twice at (0.8x), re-suspend in 10ul |
Revision as of 01:00, 30 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(.08) 5 min/PAP 10min | TSO.r06 | T20V.id2 | N2.id10 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.08) 5 min/PAP 10min/1:0.01 ATP | TSO.r06 | T20V.id2 | N2.id11 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.08) 5 min/PAP 10min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id12 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.08) 5 min/PAP 10min/1:0.1 ATP | TSO.r06 | T20V.id2 | N2.id13 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.04) 5 min/PAP 10min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id14 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.01) 5 min/PAP 10min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id15 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - no RNase3 5 min/PAP 10min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id16 | totoRNAseq |
Note: ATP Condtions:
- 1mM ATP/0mM 3'dATP
- 1mM ATP/0.01mM 3'dATP
- 1mM ATP/0.05mM 3'dATP
- 1mM ATP/0.1mM 3'dATP
Purpose
- Continuation of tests from 10-23-2013
- To try and reduce the number of PolyA reads in the totoRNAseq library that might arise from overfragmentation or overactive PAP
- Test [RNaseIII] on fragmentation and resultant post-PCR amplification
- Test termination of PAP activity to limit poly-A tail length by adding Cordycepin-ATP or 3'-ATP at different ratios to regular ATP
- Include PAP inactivation - heating
RNAseIII-Fragmented Library Preparation
- RNA
Volume UHRR 200pg/ul 0.5ul ERCC 1:10E5 0.2ul 5x Ambion PAP buffer 0.08ul 0.1uM T20V.id2 0.1ul 25mM MnCl2 0.08ul dH20 0.29ul Total 1.25ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- RNA FRAGMENTATION
Volume RNA 1.25ul RNase III 0.08ul or 0.04ul or 0.01ul dH20 0.67ul or 0.71ul or 0.74ul Total 2ul
- Incubate @ 37C 5 min
- Incubate @ 65C 10 min
- 3' Tailing
Volume fRNA 2ul 5x Ambion PAP Buffer 0.72ul 25mM MnCl2 0.32ul ATP (or mix) 0.25ul 0.4U/ul PAP 0.2ul dH2O 0.51ul
- 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 10 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
- Add 15ul beads per well
- Allow to bind 15 min - combine sets
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- 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 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:
Note: Numbers refer to:
- id10 RNaseIII (0.08U)/1mM ATM
- id11 RNaseIII (0.08U)/1mM:0.01mM ATM:3'dATP
- id12 RNaseIII (0.08U)/1mM:0.05mM ATM:3'dATP
- id13 RNaseIII (0.08U)/1mM:0.1mM ATM:3'dATP
- id14 RNaseIII (0.04U)/1mM:0.05mM ATM:3'dATP
- id15 RNaseIII (0.01U)/1mM:0.05mM ATM:3'dATP
- id16 No RNaseIII/1mM:0.05mM ATM:3'dATP
- id17 NC
Final Library Preparation
- Beads purified twice at (0.8x), re-suspend in 10ul