Blue:RNA-Seq Experiments:PAP opt: Difference between revisions
Jump to navigation
Jump to search
>B1lake |
>B1lake |
||
Line 34: | Line 34: | ||
== Purpose == | == Purpose == | ||
* | *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 | **Test reducing RNaseIII fragmentation times | ||
*Test PAP inactivation - heating | **Test PAP inactivation - heating | ||
*Test PAP | **Test termination of PAP activity to limit poly-A tail length by adding Cordycepin-ATP or 3'-ATP at different ratios to regular ATP | ||
== RNAseIII-Fragmented Library Preparation == | == RNAseIII-Fragmented Library Preparation == |
Revision as of 23:15, 21 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(1st half)/huNu(2nd half)/ERCC | 10pg - RNase3 2 min | TSO.r06 | T20V.id1-8 | N2.id1 | totoRNAseq | Read1 plus N2 barcode read | |
BL_ | UHRR(1st half)/huNu(2nd half)/ERCC | 10pg - RNase3 4 min | TSO.r06 | T20V.id9-16 | N2.id2 | totoRNAseq | Read1 plus N2 barcode read | |
BL_ | UHRR(1st half)/huNu(2nd half)/ERCC | 10pg - RNase3 6 min | TSO.r06 | T20V.id17-24 | N2.id3 | totoRNAseq | Read1 plus N2 barcode read | |
BL_ | UHRR(1st half)/huNu(2nd half)/ERCC | 10pg - RNase3 6 min - 0.01/1 3'-dATP/ATP | TSO.r06 | T20V.id25-32 | N2.id4 | totoRNAseq | Read1 plus N2 barcode read | |
BL_ | UHRR(1st half)/huNu(2nd half)/ERCC | 10pg - RNase3 6 min - 0.1/1 3'-dATP/ATP | TSO.r06 | T20V.id33-40 | N2.id5 | totoRNAseq | Read1 plus N2 barcode read | |
BL_ | UHRR(1st half)/huNu(2nd half)/ERCC | 10pg - RNase3 6 min - 0.5/1 3'-dATP/ATP | TSO.r06 | T20V.id41-48 | N2.id6 | totoRNAseq | Read1 plus N2 barcode read | |
BL_ | UHRR(1st half)/huNu(2nd half)/ERCC | 10pg - RNase3 6 min - Affymetrix PAP | TSO.r06 | T20V.id49-56 | N2.id7 | totoRNAseq | Read1 plus N2 barcode read | |
BL_ | UHRR(1st half)/huNu(2nd half)/ERCC | 10pg - RNase3 6 min - Affymetrix PAP - heat 65C 10min | TSO.r06 | T20V.id57-64 | N2.id8 | totoRNAseq | Read1 plus N2 barcode read |
Purpose
- To try and reduce the number of PolyA reads in the totoRNAseq library that might arise from overfragmentation or overactive PAP
- Test reducing RNaseIII fragmentation times
- Test PAP inactivation - heating
- Test termination of PAP activity to limit poly-A tail length by adding Cordycepin-ATP or 3'-ATP at different ratios to regular ATP
RNAseIII-Fragmented Library Preparation
- Cell Sort
Volume RNA 250pg or Single Nuclei - 1x Lysis Buffer 0.75ul Total 0.75ul
Note: add in UHRR control at this point
- Lysis
Volume RNA 0.75ul 10x RNaseIII Buffer 0.2ul 0.1uM T20V.id 0.1ul dH20 0.2ul (1250pg/ul UHRR for 250pg or 50pg/ul for 10pg) Total 1.25ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- Incubate @ 4C 10min
- RNA FRAGMENTATION
Volume RNA 1.25ul RNase III 0.08ul dH20 0.67ul Total 2ul
- Incubate @ 37C 10 min
- Add 1.1ul dH2O
- Incubate @ 65C 10 min
- 3' Tailing
Volume fRNA 3.1ul 5x RT Buffer 0.5ul (0.8ul) 1mM ATP (or mix) 0.25ul (0.1ul of 2.5mM) 0.5U/ul PAP (1:10d in H2O) 0.25ul (0.1ul of 1:4)
- Incubate @ 37C 5 min
- Transfer to Ice
- Reverse Transcription
Volume PAP RNA 4.1ul 10mM dNTP Mix 1ul 0.1uM T20V.ID 0.9ul
- Incubate @ 65C 3 min (or 10 min)
- Transfer to Ice
Volume 5xRT Buffer 1.5ul (1.2ul) 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
- 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 11ul 2x Buffer 25ul 10uM P1-STRT 1ul 10uM PCR_R_N2_id 1ul H2O 12ul 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
- Re-Amplified using ILMN_PCR_F/R:
- huNu (~30) id81 = 2ul original PCR
- UHRR (250pg) = 0.5ul original PCR
- NC
- Ran 5ul (of 50ul) on gel:
- Clearly the huNu library did not work...