Difference between revisions of "User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/26"

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span>
 
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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* Step 3, 4 - Add Experiments to Import: added '''6_aln_sorted.bam''' (H3K27me3 ChIP), and '''inputU2OS.bam''' (DNA purified from unenriched chromatin)
 
* Step 3, 4 - Add Experiments to Import: added '''6_aln_sorted.bam''' (H3K27me3 ChIP), and '''inputU2OS.bam''' (DNA purified from unenriched chromatin)
 
* Step 5 - Create binding proteins: created "H3K27me3" for 6_aln_sorted.
 
* Step 5 - Create binding proteins: created "H3K27me3" for 6_aln_sorted.
* Set control inputU2OS as "yes" for "Is control?"
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* Step 6 - Assigned H3K27me3 as protein label for 6_aln_sorted; Set control inputU2OS as "yes" for "Is control?"; Set inputU2OS as control for 6_aln_sorted.
* Set inputU2OS as control for 6_aln_sorted.
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* Step 11 - Chose BED files only (wigs take up lots of disk space)
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* Step 12 - Deselected "Directionality"
  
  

Latest revision as of 21:48, 26 September 2017

Owwnotebook icon.pngPc-TF Genomics Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png


06/26/13

  • ChIP-seq: use input results to determine enrichment of U2OS H3K27me3 signals with Seqman NGen
  • TRIzol prep: U2OS PcTF+ & mock cells



ChIP seq analysis with Input control
> Follow workflow from http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2013/01/07

Array Star analysis - general instructions

  1. Open Array Star. Windows only. This can be run on Parallels from a Mac.
  2. Click "Start Chip-Seq project..."
  3. Add Experiments to Import: Click [Add File..]
  4. Select a ###.bam file from the ChiP seq data folder (shared desktop). Click [Next >].
  5. Create binding proteins: Click [Create Binding Protein]. Binding protein label = the name of the protein that was pulled down by the antibody via ChIP. Binding site type = Unknown. Click [OK].
  6. Assign Binding Proteins to Experiments: Protein label = the appropriate binding protein (created in previous step). Click [Next >].
  7. Setup Preprocessing: Desired preprocessing = QSeq; Normalization method = none; Sequence files against which to quantify = browse to Desktop > Human Genome Ref > select all NC...gbk files.
  8. Select "Use features of type(s)" and set to "gene".
  9. Genome filtering = Discover peaks in the entire genome.
  10. Configure Advanced Options: under General, Graphs > Create Wiggle (.wig). Set Location to ChIPseq Wig Files on desktop.
  11. Configure Advanced Options: under General, Alignment Files > Create BED Alignment (.bed). Set Location to ChIPseq BED Files on desktop.
  12. Configure Advanced Options: under Peak Detection, is set to Qseq Peak Finder by default. Leave this as-is for now.
  13. Click [Next >]. Wait a while
  14. Setup Attributes and Replicates: Show only grouping attributes (default). Click [Finish].
  15. Window with Peak Table and other tabs should appear. Finished! Explore the data.

H3K27me3 enrichment in U2OS

  • Step 3, 4 - Add Experiments to Import: added 6_aln_sorted.bam (H3K27me3 ChIP), and inputU2OS.bam (DNA purified from unenriched chromatin)
  • Step 5 - Create binding proteins: created "H3K27me3" for 6_aln_sorted.
  • Step 6 - Assigned H3K27me3 as protein label for 6_aln_sorted; Set control inputU2OS as "yes" for "Is control?"; Set inputU2OS as control for 6_aln_sorted.
  • Step 11 - Chose BED files only (wigs take up lots of disk space)
  • Step 12 - Deselected "Directionality"



U2OS TRIzol prep

  1. +PcTF, plate 1-A
  2. +PcTF, plate 1-B
  3. mock, plate 4-A
  4. mock, plate 4-B

(Should provide more than enough RNA. Discarded other cultures)

  • Cells were 100% confluent
  • Discarded growth medium
  • Added 2 mL TRIzol directly to plates
  • Incubated at r.t. for ~5 min.
  • Collected lysed cells from plate with gentle scraping (pipette tip)
  • Transferred 500 μL aliquots to 2.0 mL tubes. Stored at -80°C
    • 8x PcTF+ samples
    • 8x mock samples