|
Status |
Public on Jan 01, 2012 |
Title |
Immunoprecipitated chromatin, antibody 2, replicate 2 Set 2 |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
Ramos cells, nonIP, control
|
Organism |
Homo sapiens |
Characteristics |
cell type: Ramos germinal center B cells cell type: Burkitt's lymphoma cell line antibody: none
|
Treatment protocol |
Untreated cells were used for chromatin isolation.
|
Growth protocol |
Ramos cells were grown in RPMI supplemented with 10% fetal bovine serum, non-essential amino acids, and antibiotics.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
2x10^8 cells were pelleted, washed with PBS, and lysed with hypotonic buffer on ice for 10 minutes to release the cytoplasm. Nuclei were pelleted at 4 degrees C and lysed with ice-cold nuclear lysis buffer supplemented with protease inhibitor, on ice for 10 minutes. Chromatin was sheared by sonication, using 5 pulses of 30 seconds each, separated by 1-minute rests. DNA size was analyzed on a 2% agarose gel, and appeared as a smear ranging from 0.5-1 kb.
|
Label |
Cy3
|
Label protocol |
500ng of the control and ChIP-enriched genomic DNA was heat fragmented at 95C for 30’. The heat fragmented DNA was labeled using BioPrime DNA Labeling System (Invitrogen, Carlsbad, CA) with some modifications.
|
|
|
Channel 2 |
Source name |
Ramos cells, immunoprecipitataed, test
|
Organism |
Homo sapiens |
Characteristics |
cell type: Ramos germinal center B cells cell type: Burkitt's lymphoma cell line antibody: Ab2 = EMD Biosciences #ST1099
|
Treatment protocol |
Untreated cells were used for chromatin isolation.
|
Growth protocol |
Ramos cells were grown in RPMI supplemented with 10% fetal bovine serum, non-essential amino acids, and antibiotics.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
2x10^8 cells were pelleted, washed with PBS, and lysed with hypotonic buffer on ice for 10 minutes to release the cytoplasm. Nuclei were pelleted at 4 degrees C and lysed with ice-cold nuclear lysis buffer supplemented with protease inhibitor, on ice for 10 minutes. Chromatin was sheared by sonication, using 5 pulses of 30 seconds each, separated by 1-minute rests. DNA size was analyzed on a 2% agarose gel, and appeared as a smear ranging from 0.5-1 kb.
|
Label |
Cy5
|
Label protocol |
500ng of the control and ChIP-enriched genomic DNA was heat fragmented at 95C for 30’. The heat fragmented DNA was labeled using BioPrime DNA Labeling System (Invitrogen, Carlsbad, CA) with some modifications.
|
|
|
|
Hybridization protocol |
The purified enriched and control DNA were hybridized to an Agilent 244K Human Promoter ChIP-on-chip Microarray Set (Agilent Technologies, Palo Alto, CA) using Maui AO (BioMicro Systems, Salt Lake City, UT). The microarrays were hybridized at 60º in a MAUI hybridization station on setting B (12spotMAUI) for 18 hours and then washed using Agilent aCGH wash buffers following the provided protocols.
|
Scan protocol |
Microarrays were scanned using the Agilent DNA microarray scanner, and data were extracted from images using the Agilent Feature Extraction (version 9.5.1.1) software.
|
Description |
112707_251470711232_S01_CGH-v4_91_Amgen_082406.txt
|
Data processing |
The data were extracted using the Agilent Feature Extraction (version 9.5.1.1) software using the standard Agilent protocol with Lowess normalization.
|
|
|
Submission date |
Jul 26, 2010 |
Last update date |
Jan 01, 2012 |
Contact name |
Chetan Deshpande |
E-mail(s) |
chetand@amgen.com
|
Organization name |
Amgen Inc
|
Department |
Molecular Sciences
|
Street address |
One Amgen Center Dr
|
City |
Newbury Park |
State/province |
CA |
ZIP/Postal code |
91320 |
Country |
USA |
|
|
Platform ID |
GPL4125 |
Series (2) |
GSE23170 |
ChIP-on-chip experiment from Ramos cells to analyze genome-wide CRTC2 binding sites in germinal center B cells |
GSE23171 |
CRTC2 couples genotoxic stress and plasma cell differentiation in the germinal center |
|