Blue:RNA-Seq Experiments:09102013: Difference between revisions
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| BL_ERCC_N2id1_Sept10||ERCC||1ng - RNaseIII Fragmented||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r04]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id1]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id1]||totoRNAseq||QC qPCR Analysis|| | | BL_ERCC_N2id1_Sept10||ERCC||1ng - RNaseIII Fragmented||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r04]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id1]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id1]||totoRNAseq||QC qPCR Analysis|| | ||
|- | |- | ||
| BL_ERCC_N2id2_Sept10||ERCC||1ng - Mg Fragmented||TSO.R04||T20V. | | BL_ERCC_N2id2_Sept10||ERCC||1ng - Mg Fragmented||TSO.R04||T20V.id96||N2.id2|||||| | ||
|- | |- | ||
| BL_ERCC_N2id3_Sept10||ERCC||1ng - Unfragmented||TSO.R04||T20V. | | BL_ERCC_N2id3_Sept10||ERCC||1ng - Unfragmented||TSO.R04||T20V.id96||N2.id3|||||| | ||
|- | |- | ||
| BL_ERCC_N2id4_Sept10||ERCC||1ng - RNaseIII Fragmented||TSO.R05||T20V. | | BL_ERCC_N2id4_Sept10||ERCC||1ng - RNaseIII Fragmented||TSO.R05||T20V.id96||N2.id4|||||| | ||
|- | |- | ||
| BL_ERCC_N2id5_Sept10||ERCC||1ng - Mg Fragmented||TSO.R05||T20V. | | BL_ERCC_N2id5_Sept10||ERCC||1ng - Mg Fragmented||TSO.R05||T20V.id96||N2.id5|||||| | ||
|- | |- | ||
| BL_ERCC_N2id6_Sept10||ERCC||1ng - Unfragmented||TSO.R05||T20V. | | BL_ERCC_N2id6_Sept10||ERCC||1ng - Unfragmented||TSO.R05||T20V.id96||N2.id6|||||| | ||
|- | |- | ||
| BL_ERCC_N2id7_Sept10||ERCC||1ng - RNAseIII Fragmented||Smarter TSO||T20V. | | BL_ERCC_N2id7_Sept10||ERCC||1ng - RNAseIII Fragmented||Smarter TSO||T20V.id96||N2.id7|||||| | ||
|- | |- | ||
| BL_ERCC_N2id8_Sept10||ERCC||1ng - Mg Fragmented||Smarter TSO||T20V. | | BL_ERCC_N2id8_Sept10||ERCC||1ng - Mg Fragmented||Smarter TSO||T20V.id96||N2.id8|||||| | ||
|- | |- | ||
| BL_ERCC_N2id9_Sept10||ERCC||1ng - Unfragmented||Smarter TSO||T20V. | | BL_ERCC_N2id9_Sept10||ERCC||1ng - Unfragmented||Smarter TSO||T20V.id96||N2.id9|||||| | ||
|- | |- | ||
| BL_ERCC_N2id_Sept10||ERCC||1ng - RNaseIII Fragmented||TSO.R04||Supp.T20.id1||N2.id10|||||| | | BL_ERCC_N2id_Sept10||ERCC||1ng - RNaseIII Fragmented||TSO.R04||Supp.T20.id1||N2.id10|||||| |
Revision as of 19:27, 10 September 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_ERCC_N2id1_Sept10 | ERCC | 1ng - RNaseIII Fragmented | TSO.r04 | T20V.id1 | N2.id1 | totoRNAseq | QC qPCR Analysis | |
BL_ERCC_N2id2_Sept10 | ERCC | 1ng - Mg Fragmented | TSO.R04 | T20V.id96 | N2.id2 | |||
BL_ERCC_N2id3_Sept10 | ERCC | 1ng - Unfragmented | TSO.R04 | T20V.id96 | N2.id3 | |||
BL_ERCC_N2id4_Sept10 | ERCC | 1ng - RNaseIII Fragmented | TSO.R05 | T20V.id96 | N2.id4 | |||
BL_ERCC_N2id5_Sept10 | ERCC | 1ng - Mg Fragmented | TSO.R05 | T20V.id96 | N2.id5 | |||
BL_ERCC_N2id6_Sept10 | ERCC | 1ng - Unfragmented | TSO.R05 | T20V.id96 | N2.id6 | |||
BL_ERCC_N2id7_Sept10 | ERCC | 1ng - RNAseIII Fragmented | Smarter TSO | T20V.id96 | N2.id7 | |||
BL_ERCC_N2id8_Sept10 | ERCC | 1ng - Mg Fragmented | Smarter TSO | T20V.id96 | N2.id8 | |||
BL_ERCC_N2id9_Sept10 | ERCC | 1ng - Unfragmented | Smarter TSO | T20V.id96 | N2.id9 | |||
BL_ERCC_N2id_Sept10 | ERCC | 1ng - RNaseIII Fragmented | TSO.R04 | Supp.T20.id1 | N2.id10 | |||
BL_ERCC_N2id_Sept10 | ERCC | 1ng - Mg Fragmented | TSO.R04 | Supp.T20.id1 | N2.id11 | |||
BL_ERCC_N2id_Sept10 | ERCC | 1ng - Unfragmented | TSO.R04 | Supp.T20.id1 | N2.id12 |
Purpose
- Examine efficiency of TSO primers using ERCC assay
- Examine efficiency of TSO between fragmented and unfragmented using ERCC assay
- Compare RNaseIII and Mg based fragmentation methods
Library Preparation (RNAseIII Fragmentation):
- RNA FRAGMENTATION
Volume 1ng UHRR 0.5ul ERCC 1:10E4 0.2ul 10x RNaseIII Buffer 0.2ul 1uM T20V.id1 or Supp.T20.id 0.1ul dH20 0.25ul 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 PNK_fRNA 3.1ul 5x RT Buffer 0.5ul 1mM ATP 0.25ul 0.5U/ul PAP (1:10d in H2O) 0.25ul
- Incubate @ 37C 5 min
- Transfer to Ice
Library Preparation (Mg-based Fragmentation)
- RNA FRAGMENTATION
Volume 1ng UHRR RNA 0.5ul 1:1E104 ERCC 0.2ul 5x RT buffer 1ul dH2O 0.3ul Total 2ul
- Incubate @ 94C 2 min
- Transfer to ice
- End Repair
Volume fRNA 2ul 1mM ATP 0.4ul 100mM DTT 0.1ul RNase Inhibitor 0.1ul PNK 0.5ul Total 3.1ul
- Incubate @ 37C 30 min
- 3' Tailing
Volume PNK_fRNA 3.1ul 1mM ATP 0.5ul 0.5U/ul PAP (1:10d in H2O) 0.5ul
- Incubate @ 37C 5 min
- Transfer to Ice
Reverse Transcription:
Volume PAP RNA or unfrag RNA (diluted to 3.6ul in dH2O) 4.1ul (3.6ul) 10mM dNTP Mix 1ul 1uM T20V.ID or Supp.T20.id 0.9ul
- Incubate @ 65C 3 min
- Transfer to Ice
Volume 5xRT Buffer 1.5ul (RNaseIII); 1ul + 0.5ul dH2O (Mg); 2ul (unfrag) 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
- 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
TSO
- TSO (x2)
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
- USER
- Add 1ul and incubate at 37C for 30 min
- PCR (PhusionHF)
Volume TSO Reaction 11ul 2x Buffer 25ul 10uM PB_PCR_F or PCR_Blue_Short 1ul 10uM PCR_N2_idX or PCR_Blue_Short 1ul H2O 12ul Total 50ul
98C 30 sec 98C 15s -> 58C 20s -> 72C 30s 6x 98C 10s -> 72C 20s 11x 72C, 5min
Results of Amplification
- Ran 1ul on gel:
TSO Quantification
- Comparison of Pre-amp and post-amp plots:
PCR - 5' TSO PCR - 3' T20 PCR fragment TSO+130-5' T20+130-5' ERCC-130-5' TSO+130-mid T20+130-mid ERCC-130-mid TSO+130-3' T20+130-3' ERCC-130-3'
TSO=ILMN_PCR_F; T20=ILMN_PCR_R
- Amplified 1ul TSO (diluted 5ul to 50ul) and 1ul PCR (total volume 50ul) using Sybr-Green:
Volume Library 1ul 2xSybr Buffer 12.5ul 10uM F primer 0.5ul 10uM R Primer 0.5ul dH2O 10.5ul Total 25ul
- 95C 30sec
- 95C 10s -> 60C 20s -> 72C 30s => 40X
- 72C, 5min
- Ran 5ul on gel: