User:Karmella Haynes/Notebook/PcTF Genomics/2013/01/07

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(Autocreate 2013/01/07 Entry for User:Karmella_Haynes/Notebook/PcTF_Genomics)
Current revision (16:24, 8 January 2013) (view source)
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==mm/dd/yy==
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==01/07/13==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
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* Array Star analysis
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----
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'''Line item 1'''<br>
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'''Array Star analysis'''<br>
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> Samples
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{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
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# Open '''Array Star'''. ''Windows only. This can be run on Parallels from a Mac.''
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|-valign="top"
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# Click "Start Chip-Seq project..."
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| <u>Reagent</u> || <u>Volume</u>  
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# Add Experiments to Import: Click [Add File..]
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|-
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# Select a ###.bam file from the ChiP seq data folder (shared desktop). Click [Next >].
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| reagent 1 || # μL
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# 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].
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|-
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# Assign Binding Proteins to Experiments: Protein label = the appropriate binding protein (created in previous step). Click [Next >].
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| reagent 2 || #
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# 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.
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|-
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## Select "Use features of type(s)" and set to "gene".
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| reagent 3 || #
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## Genome filtering = Discover peaks in the entire genome.
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|-
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## Configure Advanced Options: under General, Graphs > Create Wiggle (.wig). Set Location to ChIPseq Wig Files on desktop.
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| reagent 4 || #
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## Configure Advanced Options: under General, Alignment Files > Create BED Alignment (.bed). Set Location to ChIPseq BED Files on desktop.
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|-
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## Configure Advanced Options: under Peak Detection, is set to Qseq Peak Finder by default. Leave this as-is for now.
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| dH<sub>2</sub>O || #
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## Click [Next >]. Wait a while
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|-
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# Setup Attributes and Replicates: Show only grouping attributes (default). Click [Finish].
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| &nbsp; || # μL
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# Window with Peak Table and other tabs should appear. Finished! Explore the data.
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|}
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--> Reaction conditions
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'''PcTF vs. H3K27me3'''
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* Add Experiments to Import: added  1_aln_sorted.bam (PcTF), 2_aln_sorted.bam (PcTF mock), 6_aln_sorted.bam (H3K27me3), and 7_aln_sorted.bam (H3K27me3 mock)
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* Create binding proteins: created "PcTF" for 1_aln_sorted, and "H3K27me3" for 6_aln_sorted.
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* Set control 2_ and 7_aln_sorted as "yes" for "Is control?"
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* Set 2_aln_sorted as control for 1_aln_sorted. Set 7_aln_sorted as control for 6_aln_sorted.
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** <font color="red">This analysis required too much disk space</font>
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* Started over, but only used '''chromosomes 1-3''' as reference templates (trying a few at a time).
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** This worked
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Results/ Notes:
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* Peak Table: shows very few hits for PcTF, many more for H3K27me3, no overlap!
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* Display wig file on UCSC Genome browser online - add file from "ChIPseq Wig Files" folder as a custom track; wig file shows raw data histogram
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** 1_aln_sorted.wig, 6_aln_sorted.wig
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* Display BED file on UCSC Genome browser online - add file from "ChIPseq BED Files" folder as a custom track; BED file shows
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** 1_aln_sorted.bed, 6_aln_sorted.bed

Current revision

Pc-TF Genomics Main project page
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01/07/13

  • Array Star analysis

Array Star analysis

  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.
    1. Select "Use features of type(s)" and set to "gene".
    2. Genome filtering = Discover peaks in the entire genome.
    3. Configure Advanced Options: under General, Graphs > Create Wiggle (.wig). Set Location to ChIPseq Wig Files on desktop.
    4. Configure Advanced Options: under General, Alignment Files > Create BED Alignment (.bed). Set Location to ChIPseq BED Files on desktop.
    5. Configure Advanced Options: under Peak Detection, is set to Qseq Peak Finder by default. Leave this as-is for now.
    6. Click [Next >]. Wait a while
  8. Setup Attributes and Replicates: Show only grouping attributes (default). Click [Finish].
  9. Window with Peak Table and other tabs should appear. Finished! Explore the data.


PcTF vs. H3K27me3

  • Add Experiments to Import: added 1_aln_sorted.bam (PcTF), 2_aln_sorted.bam (PcTF mock), 6_aln_sorted.bam (H3K27me3), and 7_aln_sorted.bam (H3K27me3 mock)
  • Create binding proteins: created "PcTF" for 1_aln_sorted, and "H3K27me3" for 6_aln_sorted.
  • Set control 2_ and 7_aln_sorted as "yes" for "Is control?"
  • Set 2_aln_sorted as control for 1_aln_sorted. Set 7_aln_sorted as control for 6_aln_sorted.
    • This analysis required too much disk space
  • Started over, but only used chromosomes 1-3 as reference templates (trying a few at a time).
    • This worked


Results/ Notes:

  • Peak Table: shows very few hits for PcTF, many more for H3K27me3, no overlap!
  • Display wig file on UCSC Genome browser online - add file from "ChIPseq Wig Files" folder as a custom track; wig file shows raw data histogram
    • 1_aln_sorted.wig, 6_aln_sorted.wig
  • Display BED file on UCSC Genome browser online - add file from "ChIPseq BED Files" folder as a custom track; BED file shows
    • 1_aln_sorted.bed, 6_aln_sorted.bed



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