Blue:RNA-Seq Experiments:10032013: Difference between revisions
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>B1lake |
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| align="center" style="background:#f0f0f0;"|'''Library Conc''' | | align="center" style="background:#f0f0f0;"|'''Library Conc''' | ||
|- | |- | ||
| | | BL_LCM_10Cell_N2id1_Oct03||Mouse Pyramidal|| 10 cell(C1) - water 70C 5min||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id1]||totoRNAseq||Read1 plus N2 barcode read || | ||
|- | |- | ||
| | | BL_LCM_10Cell_N2id2_Oct03||Mouse Pyramidal|| 10 cell(C3) - water 70C 5min||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id2]||totoRNAseq||Read1 plus N2 barcode read || | ||
|- | |- | ||
| | | BL_LCM_10Cell_N2id3_Oct03||Mouse Pyramidal|| 10 cell(T1) - Tritonx100 55C 30min||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id3]||totoRNAseq||Read1 plus N2 barcode read || | ||
|- | |- | ||
| | | BL_LCM_10Cell_N2id4_Oct03||Mouse Pyramidal|| 10 cell(T4) - Tritonx100 55C 30min||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id4]||totoRNAseq||Read1 plus N2 barcode read || | ||
|- | |- | ||
| | | BL_LCM_10Cell_N2id5_||Mouse Pyramidal|| 10 cell(1) - Extraction||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id5]||totoRNAseq||Read1 plus N2 barcode read || | ||
|- | |- | ||
| | | BL_LCM_5Cell_N2id6_||Mouse Pyramidal|| 5 cell(1) - Extraction||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id6]||totoRNAseq||Read1 plus N2 barcode read || | ||
|- | |- | ||
| | | BL_LCM_100pgHC_N2id7_Oct03||Mouse Pyramidal|| Handcut(2) - Extraction||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id7]||totoRNAseq||Read1 plus N2 barcode read || | ||
|- | |- | ||
| | | BL_LCM_100pgHC_N2id8_Oct03||Mouse Pyramidal|| Handcut(2) - Extraction||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence TSO.r06]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence T20V.id2]||[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_3.27_primer_sequence N2.id8]||totoRNAseq||Read1 plus N2 barcode read || | ||
|- | |- | ||
|} | |} | ||
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== Purpose == | == Purpose == | ||
*Generate totoRNAseq libraries using RNaseIII digestion on LCM collected samples of | *Generate totoRNAseq libraries using RNaseIII digestion on LCM collected samples of mouse brain pyramidal neurons | ||
*Use LCM samples that have been extracted for RNA as control | *Use LCM samples that have been extracted for RNA as control | ||
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*Dilute handcut to 133pg/ul (1:10 dilution) | *Dilute handcut to 133pg/ul (1:10 dilution) | ||
*Samples collected in 10ul water or Triton-X100 | *Samples collected in 10ul water or Triton-X100 | ||
*Vacuspin to bring volume to | *Vacuspin to bring volume to 1.0ul | ||
Note: | |||
:*15 min at RT | |||
:**plus 5min at 30C for Extracted (5 cell actually dried out - added 1ul water to try and re-hydrate) | |||
:**plus 7min for C1,C3,T1,T4 | |||
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|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
|RNA|||||||||| | |RNA||||||||||1.0ul | ||
|- | |- | ||
|ERCC 1:10E5||||||||||0.2ul | |ERCC 1:10E5||||||||||0.2ul | ||
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|10x RNaseIII Buffer||||||||||0.2ul | |10x RNaseIII Buffer||||||||||0.2ul | ||
|- | |- | ||
|0.1uM T20V. | |0.1uM T20V.id2||||||||||0.1ul | ||
|- | |- | ||
|Total||||||||||1. | |Total||||||||||1.5ul | ||
|- | |- | ||
|} | |} | ||
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|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
|RNA||||||||||1. | |RNA||||||||||1.5ul | ||
|- | |- | ||
|RNase III||||||||||0.08ul | |RNase III||||||||||0.08ul | ||
|- | |- | ||
|dH20||||||||||0. | |dH20||||||||||0.42ul | ||
|- | |- | ||
|Total||||||||||2ul | |Total||||||||||2ul | ||
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|10mM dNTP Mix||||||||||1ul | |10mM dNTP Mix||||||||||1ul | ||
|- | |- | ||
|0.1uM T20V. | |0.1uM T20V.ID2 ||||||||||0.9ul | ||
|- | |- | ||
|} | |} | ||
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|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
|TSO Reaction|||||||||| | |TSO Reaction||||||||||10ul | ||
|- | |- | ||
|2x Buffer||||||||||25ul | |2x Buffer||||||||||25ul | ||
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|10uM PCR_R_N2_id||||||||||1ul | |10uM PCR_R_N2_id||||||||||1ul | ||
|- | |- | ||
|H2O|||||||||| | |H2O||||||||||13ul | ||
|- | |- | ||
|Total||||||||||50ul | |Total||||||||||50ul | ||
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*Run 5ul on gel: | *Run 5ul on gel: | ||
[[File:10032013_PCR1_gel1.jpg]] | |||
== Final Library Preparation == | == Final Library Preparation == | ||
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*Beads purify again at 0.8x (if primer dimer band is apparent) | *Beads purify again at 0.8x (if primer dimer band is apparent) | ||
*Resuspend beads in 10ul | *Resuspend beads in 10ul | ||
*Run | *Run 1ul on gel: | ||
[[File:10032013_PCR1_gel2.jpg]] |
Latest revision as of 21:03, 10 October 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_LCM_10Cell_N2id1_Oct03 | Mouse Pyramidal | 10 cell(C1) - water 70C 5min | TSO.r06 | T20V.id2 | N2.id1 | totoRNAseq | Read1 plus N2 barcode read | |
BL_LCM_10Cell_N2id2_Oct03 | Mouse Pyramidal | 10 cell(C3) - water 70C 5min | TSO.r06 | T20V.id2 | N2.id2 | totoRNAseq | Read1 plus N2 barcode read | |
BL_LCM_10Cell_N2id3_Oct03 | Mouse Pyramidal | 10 cell(T1) - Tritonx100 55C 30min | TSO.r06 | T20V.id2 | N2.id3 | totoRNAseq | Read1 plus N2 barcode read | |
BL_LCM_10Cell_N2id4_Oct03 | Mouse Pyramidal | 10 cell(T4) - Tritonx100 55C 30min | TSO.r06 | T20V.id2 | N2.id4 | totoRNAseq | Read1 plus N2 barcode read | |
BL_LCM_10Cell_N2id5_ | Mouse Pyramidal | 10 cell(1) - Extraction | TSO.r06 | T20V.id2 | N2.id5 | totoRNAseq | Read1 plus N2 barcode read | |
BL_LCM_5Cell_N2id6_ | Mouse Pyramidal | 5 cell(1) - Extraction | TSO.r06 | T20V.id2 | N2.id6 | totoRNAseq | Read1 plus N2 barcode read | |
BL_LCM_100pgHC_N2id7_Oct03 | Mouse Pyramidal | Handcut(2) - Extraction | TSO.r06 | T20V.id2 | N2.id7 | totoRNAseq | Read1 plus N2 barcode read | |
BL_LCM_100pgHC_N2id8_Oct03 | Mouse Pyramidal | Handcut(2) - Extraction | TSO.r06 | T20V.id2 | N2.id8 | totoRNAseq | Read1 plus N2 barcode read |
LCM samples collected 10022013 (handcut 2 1.4ng/ul)
Purpose[edit]
- Generate totoRNAseq libraries using RNaseIII digestion on LCM collected samples of mouse brain pyramidal neurons
- Use LCM samples that have been extracted for RNA as control
RNAseIII-Fragmented Library Preparation[edit]
- Sample Prep
- Dilute handcut to 133pg/ul (1:10 dilution)
- Samples collected in 10ul water or Triton-X100
- Vacuspin to bring volume to 1.0ul
Note:
- 15 min at RT
- plus 5min at 30C for Extracted (5 cell actually dried out - added 1ul water to try and re-hydrate)
- plus 7min for C1,C3,T1,T4
- 15 min at RT
Volume RNA 1.0ul ERCC 1:10E5 0.2ul 10x RNaseIII Buffer 0.2ul 0.1uM T20V.id2 0.1ul Total 1.5ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1min
- Incubate @ 4C 1min
- RNA FRAGMENTATION
Volume RNA 1.5ul RNase III 0.08ul dH20 0.42ul 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 1mM ATP 0.25ul 0.5U/ul PAP (1:10d in H2O) 0.25ul
- Incubate @ 37C 5 min
- Transfer to Ice
- Reverse Transcription
Volume PAP RNA 4.1ul 10mM dNTP Mix 1ul 0.1uM T20V.ID2 0.9ul
- Incubate @ 65C 3 min
- Transfer to Ice
Volume 5xRT Buffer 1.5ul 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 - vacuspin 5 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 12x 72C, 5min
Results of Amplification[edit]
- Run 5ul on gel:
Final Library Preparation[edit]
- Beads purify (0.8x), re-suspend in 50ul
- Beads purify again at 0.8x (if primer dimer band is apparent)
- Resuspend beads in 10ul
- Run 1ul on gel: