Blue:RNA-Seq Experiments:12032013: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''Library Conc''' | | align="center" style="background:#f0f0f0;"|'''Library Conc''' | ||
|- | |- | ||
| | | BL_totoRNAseq_1ngUHRR_Dec03||UHRR/ERCC||1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id81-82||totoRNAseq|||| | ||
|- | |- | ||
| | | ||UHRR/ERCC||100pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id83-84||totoRNAseq|||| | ||
|- | |- | ||
| | | ||UHRR/ERCC||10pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id385-86||totoRNAseq|||| | ||
|- | |- | ||
| | | ||UHRR/ERCC||1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r07||T20VN_PE_R||N2.id87-88||totoRNAseq|||| | ||
|- | |- | ||
| | | ||UHRR/ERCC||1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step||TSO.r06||T20VN_PE_R||N2.id89-90||totoRNAseq|||| | ||
|- | |- | ||
| | | ||UHRR/ERCC||100pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step||TSO.r06||T20VN_PE_R||N2.id91-92||totoRNAseq|||| | ||
|- | |- | ||
| | | ||UHRR/ERCC||10pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step||TSO.r06||T20VN_PE_R||N2.id93-94||totoRNAseq|||| | ||
|- | |- | ||
| | | ||UHRR/ERCC||1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step||TSO.r07||T20VN_PE_R||N2.id95-96||totoRNAseq|||| | ||
|- | |- | ||
|} | |} | ||
Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence] | |||
== Purpose == | == Purpose == | ||
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*Beads purified twice at (0.8x), re-suspend in 10ul | *Beads purified twice at (0.8x), re-suspend in 10ul | ||
See [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNA-Seq_Experiments:12062013#Final_Library_Preparation 12062013] for gel image of final library (1ng) |
Latest revision as of 01:28, 13 December 2013
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_totoRNAseq_1ngUHRR_Dec03 | UHRR/ERCC | 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP | TSO.r06 | T20VN_PE_R | N2.id81-82 | totoRNAseq | ||
UHRR/ERCC | 100pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP | TSO.r06 | T20VN_PE_R | N2.id83-84 | totoRNAseq | |||
UHRR/ERCC | 10pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP | TSO.r06 | T20VN_PE_R | N2.id385-86 | totoRNAseq | |||
UHRR/ERCC | 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP | TSO.r07 | T20VN_PE_R | N2.id87-88 | totoRNAseq | |||
UHRR/ERCC | 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step | TSO.r06 | T20VN_PE_R | N2.id89-90 | totoRNAseq | |||
UHRR/ERCC | 100pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step | TSO.r06 | T20VN_PE_R | N2.id91-92 | totoRNAseq | |||
UHRR/ERCC | 10pg - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step | TSO.r06 | T20VN_PE_R | N2.id93-94 | totoRNAseq | |||
UHRR/ERCC | 1ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/One step | TSO.r07 | T20VN_PE_R | N2.id95-96 | totoRNAseq |
Primer sequences [1]
Purpose[edit]
- Test efficiency of Affymetrix PAP to generate libraries from decreasing amounts of RNA from 1ng down to 10pg
- Use ERCCs to determine level of transcript amplification between libraries - ERCC amplification using previous version of the totoRNAseq protocol from 09-27-13 showed progressively lower ERCC quantities in the final libraries from 100pg and 10pg UHRR
RNAseIII-Fragmented Library Preparation[edit]
- RNA
Volume UHRR 2ng/ul; 0.2ng/ul; 0.02ng/ul 0.5ul ERCC 1:10E4; 1:10E5; 1:10E6 0.2ul 0.1uM T20VN 0.1ul dH2O 0.2ul Total 1ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1 min
Volume 10x RNaseIII buffer 0.2ul RNase III 0.1ul dH20 0.7ul Total 2ul
- Incubate @ 37C 5 min
- 3' Tailing
Volume fRNA 2ul 5x RT buffer 0.5ul 1mM ATP 0.5ul 600U/ul Affymetrix PAP 0.2ul 40U/ul Rnase Inhibitor 0.2ul dH2O 0.6ul Total 4ul
- Incubate @ 37C 10 min
- Add 1ul of 0.1uM T20VN [for 1-step: 0.1ul 1mM T20VN + 0.9ul 10uM TSO]
- Reverse Transcription
Volume PAP RNA 5ul 5xRT Buffer 1.5ul 10mM dNTP Mix 1ul 100mM DTT 0.25ul Betaine (5M) 2ul Smarter MMLV (200U/ul) 0.25ul Total 10ul
- Incubate @ 42C 30 min
- Beads Purification (2-step only)
- Add 15ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin to 3ul
- TSO Reaction (2-step only)
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 (1ng) 12x (100pg) 15x (10pg) 72C, 5min
Results of Amplification[edit]
- Run 5ul on gel:
Results: Can see that the Affymetrix PAP protocol loses efficiency with decreasing RNA - may not be sensitive enough. R07 on 1ng looks very poor - too inefficient. 1-step does not appear to be an option, r07 in this context does not seem to be affected by PAP, but clearly works poorly for template switching.
Final Library Preparation[edit]
- Beads purified twice at (0.8x), re-suspend in 10ul
See 12062013 for gel image of final library (1ng)