User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/26: Difference between revisions
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* | * 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'''<br> | ||
> | > 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'''<br> | ||
# +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 | |||
Revision as of 21:14, 27 June 2013
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06/26/13
ChIP seq analysis with Input control Array Star analysis - general instructions
H3K27me3 enrichment in U2OS
U2OS TRIzol prep
(Should provide more than enough RNA. Discarded other cultures)
|