Blue:RNA-Seq Experiments:10302013: Difference between revisions
Jump to navigation
Jump to search
>B1lake |
>B1lake |
||
Line 267: | Line 267: | ||
Result: RNaseIII does not seem to | Result: RNaseIII concentration does not seem to matter... fragmentation is occurring anyway, indicating 65C enzyme inactivation steps are degrading the RNA. Need to re-optimize fragmentation step. Like there are two issues: | ||
#RNase III is still not working optimally and the majority of the fragmentation is occurring from Mn-dependent fragmentation | |||
#RNase III is fine, but already fragmented RNA is becoming further fragmented (prior to or after PAP - either way reducing poly(A)-tailed fragments for RT) | |||
Need to ensure RNase III is working and reduce opportunities for chemically induced fragmentation! | |||
== 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 21:32, 31 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(.08U) 5 min/PAP 10min | TSO.r06 | T20V.id2 | N2.id81 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.008U) 5 min/PAP 10min | TSO.r06 | T20V.id2 | N2.id82 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.001U) 5 min/PAP 10min | TSO.r06 | T20V.id2 | N2.id83 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(0U) 5 min/PAP 10min | TSO.r06 | T20V.id2 | N2.id84 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.08U) 5 min/PAP 30min | TSO.r06 | T20V.id2 | N2.id85 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.008U) 5 min/PAP 30min | TSO.r06 | T20V.id2 | N2.id86 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.001U) 5 min/PAP 30min | TSO.r06 | T20V.id2 | N2.id97 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(0U) 5 min/PAP 30min | TSO.r06 | T20V.id2 | N2.id88 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.08U) 5 min/PAP 10min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id89 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.008U) 5 min/PAP 10min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id90 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.001U) 5 min/PAP 10min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id91 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(0U) 5 min/PAP 10min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id92 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.08U) 5 min/PAP 30min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id93 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.008U) 5 min/PAP 30min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id94 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.001U) 5 min/PAP 30min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id95 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(0U) 5 min/PAP 30min/1:0.05 ATP | TSO.r06 | T20V.id2 | N2.id96 | totoRNAseq |
Note: ATP Condtions:
- 1mM ATP/0mM 3'dATP
- 1mM ATP/0.05mM 3'dATP
Purpose
- Continuation of tests from 10-23-2013 and 10-29-2013
- To try and reduce the number of PolyA reads in the totoRNAseq library that might arise from overfragmentation or overactive PAP
- Reduce [RNaseIII] to get less or no fragmentation and and see how this affects the resultant post-PCR amplification
- 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?
- 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 0.008ul or 0.001ul or 0ul dH20 up to 0.75ul 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 or 30 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 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:
Result: RNaseIII concentration does not seem to matter... fragmentation is occurring anyway, indicating 65C enzyme inactivation steps are degrading the RNA. Need to re-optimize fragmentation step. Like there are two issues:
- RNase III is still not working optimally and the majority of the fragmentation is occurring from Mn-dependent fragmentation
- RNase III is fine, but already fragmented RNA is becoming further fragmented (prior to or after PAP - either way reducing poly(A)-tailed fragments for RT)
Need to ensure RNase III is working and reduce opportunities for chemically induced fragmentation!
Final Library Preparation
- Beads purified twice at (0.8x), re-suspend in 10ul