 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 13, 2010 |
| Title |
ChIP of bio-Sall4a cells |
| Sample type |
genomic |
| |
|
| Source name |
ChIP of bio-Sall4a cells vs Input
|
| Organism |
Mus musculus |
| Characteristics |
cell type: J1 derived ES cells
experiment: ChIP with streptavidin
|
| Treatment protocol |
No treatment
|
| Growth protocol |
ES cells were grown on gelatin adapted TC plates (in the absence of feeders) in the presence of LIF and standard ES cell media till confluent
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP reactions were performed similar to (Kim J. et al, Cell, 2008). Briefly, ES cells harboring either BirA alone,Sall4a, or Sall4b were crosslinked for 10 min in 1% formaldehyde which was terminated by the addition of 125mM glycine. Cells were washed, collected, and then resuspended in ChIP dilution buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris-CL pH8.1, 150 mM NaCl and protease inhibitors). Cells were fragmented by sonication and was confirmed to have an average size of approximately 0.5-1kb by agarose gel electrophoresis. The samples were centrifuged at 40C for 10min and the supernant collected and pre-cleared with pre-washed protein A beads (Roche) at 40C for 60 min with rotation. The beads were pelleted by centrifugation and the supernant was incubated overnight at 40C with prewashed Dynabead Ò MyOneä Streptavidin T1 beads (Invitrogen). A sample of the pre-cleared supernant was saved as genomic control from the J1 ES cells expressing BirA alone. DNA absorbed beads were washed with Buffer I (2% SDS) twice, Buffer II (0.1% Deoxycholate, 1% Triton X-100, 1mM EDTA, 1mM HEPES pH7.5, 500 mM NaCl) once, Buffer III (250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA, 10 mM Tris-Cl pH 8.1) once, and TE (10 mM Tris pH8.1, 1 mM EDTA) twice. All washes were 10 min at room temperature with agitation, and beads were pelleted using magnetic separation. SDS elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1) was added and the DNA was eluted off and de-crosslinked overnight at 650C. The samples was treated with RNase A and Proteinase K, extracted with phenol:chloroform:isoamyl alcohol and precipitated. Samples were then used as either a substrate in qPCR or were amplified for hybridization to microarray. ChIP samples were amplified by LM-PCR as described in (Lupien et al., 2008) DNA was subsequently fragmented by DNase I treatment and biotin labeled per manufacturer?s instructions (www.affymetrix.com/support/downloads/manuals/chromatin_immun_ChIP.pdf).
|
| Label |
biotin
|
| Label protocol |
Fragmented DNA was labeled with biotin-N6-ddATP by terminal deoxynucleiotidyl transferase.
|
| |
|
| Hybridization protocol |
About 2-4 ug of labeled DNA were hybridized for 16 hr at 45C on tiling arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
|
| Description |
Affymetrix Mouse Promoter 1.0R array
|
| Data processing |
Model Based Analysis of Tiling Arrays (MAT) was used to extract peaks based upon default parameters, using mm8 build
The three biological replicates were pooled for each type of sample (Input, Bir A ChIP, Sall4a ChIP, Sall4b ChIP) and then compared (BirA ChIP vs. Input= Bir A; Sall4a ChIP vs Input= Sall4a; Sall4b ChIP s Input= Sall4b) and .bed and .bar files generated by MAT with a p-value set for 10^-4.
|
| |
|
| Submission date |
Mar 25, 2010 |
| Last update date |
Sep 13, 2010 |
| Contact name |
Sridhar Rao |
| E-mail(s) |
rao@bloodgroup.tch.harvard.edu
|
| Organization name |
Dana Farber Cancer Institute and Children's Hospital, Boston
|
| Department |
Pediatric Oncology
|
| Lab |
Karp Bldg, 7th floor
|
| Street address |
1 Blackfan Circle
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL5811 |
| Series (2) |
| GSE21055 |
Differential Role of Sall4 isoforms in ES cell pluripotency: ChIP-chip |
| GSE21056 |
Differential roles of Sall4 isoforms in ES cell pluripotency |
|
| Supplementary file |
Size |
Download |
File type/resource |
| GSM526869_Sall4a.bar.gz |
19.8 Mb |
(ftp)(http) |
BAR |
| GSM526869_Sall4a.bed.gz |
65.6 Kb |
(ftp)(http) |
BED |
| GSM526869_Sall4a1.CEL.gz |
17.5 Mb |
(ftp)(http) |
CEL |
| GSM526869_Sall4a2.CEL.gz |
18.5 Mb |
(ftp)(http) |
CEL |
| GSM526869_Sall4a3.CEL.gz |
18.0 Mb |
(ftp)(http) |
CEL |
| Processed data provided as supplementary file |
|
|
|
|
 |
