Array Star analysis
- Open Array Star. Windows only. This can be run on Parallels from a Mac.
- Click "Start Chip-Seq project..."
- Add Experiments to Import: Click [Add File..]
- Select a ###.bam file from the ChiP seq data folder (shared desktop). Click [Next >].
- 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].
- Assign Binding Proteins to Experiments: Protein label = the appropriate binding protein (created in previous step). Click [Next >].
- 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.
- Select "Use features of type(s)" and set to "gene".
- Genome filtering = Discover peaks in the entire genome.
- Configure Advanced Options: under General, Graphs > Create Wiggle (.wig). Set Location to ChIPseq Wig Files on desktop.
- Configure Advanced Options: under General, Alignment Files > Create BED Alignment (.bed). Set Location to ChIPseq BED Files on desktop.
- Configure Advanced Options: under Peak Detection, is set to Qseq Peak Finder by default. Leave this as-is for now.
- Click [Next >]. Wait a while
- Setup Attributes and Replicates: Show only grouping attributes (default). Click [Finish].
- 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).
- 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