Blue:RNA-Seq Experiments:10282013
Jump to navigation
Jump to search
RNA-Seq Library Information[edit]
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 2.5 min/PAP 5min | TSO.r06 | T20V.id1-8 | N2.id1 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3 2.5 min/PAP 10min | TSO.r06 | T20V.id9-16 | N2.id2 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3 2.5 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:
- 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[edit]
- 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[edit]
- RNA
Volume UHRR 200pg/ul 0.5ul ERCC 1:10E5 0.2ul 5x Ambion PAP buffer 0.08ul 0.1uM T20V.id 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 dH20 0.67ul Total 2ul
- Incubate @ 37C 2-10 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 5-30 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 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 9x 72C, 5min
Results of Amplification[edit]
- Run 5ul on gel:
Note: Looks like the RNaseIII might be over fragmenting to generate pooling of small fragments around 200bp. Increasing PAP time might be causing the shift of these up to HMW smear. This would indicate that over fragmentation followed by over polyA tailing would generate the majority of the smear. Need to sequence to know for sure. Need to repeat and separate out the ATP conditions so that each can be observed individually...
Final Library Preparation[edit]
- Beads purified twice at (0.8x), re-suspend in 10ul