User:Karmella Haynes/Notebook/PcTF Genomics/2013/06/26

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(06/26/13)
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(06/26/13)
 
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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?"
+
* 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.
+
* Step 11 - Chose BED files only (wigs take up lots of disk space)
 +
* Step 12 - Deselected "Directionality"
Line 45: Line 46:
'''U2OS TRIzol prep'''<br>
'''U2OS TRIzol prep'''<br>
-
# SK-N-SH, 1, +DNA
+
# +PcTF, plate 1-A
-
# SK-N-SH, 2, (-)
+
# +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
* Discarded growth medium
-
* Added 5 mL TRIzol directly to plates (500 mL per ~1x10<sup>6</sup> cells)
+
* Added 2 mL TRIzol directly to plates
* Incubated at r.t. for ~5 min.
* Incubated at r.t. for ~5 min.
-
* Collected lysed cells from plate with gentle scraping (pipette tip) and washing
+
* Collected lysed cells from plate with gentle scraping (pipette tip)
-
* Transferred 10x 500 μL aliquots to 2.0 mL tubes. Stored at -80°C
+
* Transferred 500 μL aliquots to 2.0 mL tubes. Stored at -80°C
 +
** 8x PcTF+ samples
 +
** 8x mock samples

Current revision

Pc-TF Genomics Main project page
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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



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