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