Blue:RNA-Seq Experiments:11012013
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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(.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.id82 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.001U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP | TSO.r06 | T20V.id2 | N2.id83 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(0U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min ATP | TSO.r06 | T20V.id2 | N2.id84 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.1U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP | TSO.r06 | T20V.id2 | N2.id85 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.01U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP | TSO.r06 | T20V.id2 | N2.id86 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(.001U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP | TSO.r06 | T20V.id2 | N2.id87 | totoRNAseq | ||
BL_ | UHRR/ERCC | 100pg - RNase3(0U) 1mM MnCl2 inactivated with Rnase Inhib/PAP 10min 3'dATP | TSO.r06 | T20V.id2 | N2.id88 | totoRNAseq |
Note: ATP Condtions:
- 1mM ATP/0mM 3'dATP
- 1mM ATP/0.05mM 3'dATP
Note: All conditions repeated for 2mM MnCl2 (N2.id89-96)
Purpose[edit]
- 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 RnaseIII inactivation with RNase Inhibitor
- Eliminate all RNA heating steps involving buffer
- 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?
RNAseIII-Fragmented Library Preparation[edit]
- RNA
Volume UHRR 200pg/ul 0.5ul ERCC 1:10E5 0.2ul 0.1uM T20V.id2 0.1ul 5x Ambion PAP buffer 0.08ul dH20 0.29ul Total 1.17ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- RNA FRAGMENTATION
Volume RNA 1.17ul 25mM MnCl2 0.08ul RNase III 0.1ul or 0.01ul or 0.001ul or 0ul dH20 up to 0.83ul Total 2ul
- Incubate @ 37C 5 min
- 3' Tailing
Volume fRNA 2ul 5x Ambion PAP Buffer 0.72ul 25mM MnCl2 0.32ul ATP (or mix) 0.25ul 2U/ul PAP 0.2ul 40U/ul Rnase Inhibitor 0.2ul dH2O 0.31ul
- Incubate @ 37C 10 min
- Transfer to Ice
- Reverse Transcription
Volume PAP RNA 4ul 10mM dNTP Mix 1ul 0.2uM T20V.ID 0.5ul 5xRT Buffer 2ul 100mM DTT 0.25ul Betaine (5M) 2ul Smarter MMLV (200U/ul) 0.25ul 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[edit]
- Run 5ul on gel:
Results:
- RNaseIII does not seem to be active in the PAP buffer conditions - neither 1mM nor 2mM MnCl2
- Elimination of heat inactivation steps seems to prevent over-fragmentation
Final Library Preparation[edit]
- Beads purified twice at (0.8x), re-suspend in 10ul