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Sample GSM611074 Query DataSets for GSM611074
Status Public on Jan 21, 2011
Title 01_Pol2_rep1
Sample type genomic
 
Channel 1
Source name Pol2 ChIP DNA from wild-type splenic B cells induced to undergo CSR
Organism Mus musculus
Characteristics genotype: wild-type
cell type: cultured splenic B cells
strain: C57BL/6
chip antibody: Pol2
chip antibody manufacturer: Upstate
chip antibody catalog #: 05-623
chip antibody lot #: DAM1661037
Treatment protocol Cultures induced for 2 days with 50 μg/ml LPS, 100 ng/ml human BLyS, and 25 ng/ml anti–δ-dextran
Growth protocol Single-cell suspensions were prepared from spleens of 6 to 12 week old mice
Extracted molecule genomic DNA
Extraction protocol Cells were washed in PBS and fixed for 10 min at 37°C with 1% formaldehyde. Fixation was quenched by incubating with glycine at a final concentration of 125 mM for 5 minutes at room temperature. Fixed cells were then washed 3 times with PBS and the dried pellet was stored at -80°C. To perform ChIP, the pellet was thawed in Lysis buffer as per the Upstate protocol in the presence of protease and phosphatase inhibitors, and then sonicated 20 times for 10 sec bursts using a VibraCell (Sonics) sonicator at a medium setting. Aliquots containing 2 million cell equivalents of lysate were incubated overnight at 4°C with a mixture of Protein A/Protein G Dynabeads (Invitrogen) coupled with specific antibody. Beads were then washed according to the Upstate protocol and DNA was de-crosslinked and eluted by overnight incubation in elution buffer at 65°C. Eluted DNA and an aliquot of the input DNA were treated with Proteinase K and RNase A (final concentrations of 125 µg/ml) at 55°C overnight. DNA was precipitated in ethanol after phenol/chloroform extraction and resuspended in Tris-EDTA (10/0.1) pH 7 buffer.
Label Cy5
Label protocol DNA samples were amplified using the WGA2 kit (Sigma Corp) according to the provided protocol, except the first fragmentation step was omitted. ChIP samples were amplified undiluted, whereas input control samples were amplified at a starting concentration of 1-2 ng/µl. After amplification, IP and input DNAs were labeled with Cy5 and Cy3 nonamers, respectively (TriLink Biotechnologies) using random primers and linear amplification with Klenow fragment of E. coli DNA Polymerase I (eBioscience Biotechnologies).
 
Channel 2
Source name input DNA from wild-type splenic B cells induced to undergo CSR
Organism Mus musculus
Characteristics genotype: wild-type
cell type: cultured splenic B cells
strain: C57BL/6
chip antibody: none, input DNA
Treatment protocol Cultures were induced for 2 days with 50 μg/ml LPS, 100 ng/ml human BLyS, and 25 ng/ml anti–δ-dextran
Growth protocol Single-cell suspensions were prepared from spleens of 6 to 12 week old mice
Extracted molecule genomic DNA
Extraction protocol Cells were washed in PBS and fixed for 10 min at 37°C with 1% formaldehyde. Fixation was quenched by incubating with glycine at a final concentration of 125 mM for 5 minutes at room temperature. Fixed cells were then washed 3 times with PBS and the dried pellet was stored at -80°C. To perform ChIP, the pellet was thawed in Lysis buffer as per the Upstate protocol in the presence of protease and phosphatase inhibitors, and then sonicated 20 times for 10 sec bursts using a VibraCell (Sonics) sonicator at a medium setting. Aliquots containing 2 million cell equivalents of lysate were incubated overnight at 4°C with a mixture of Protein A/Protein G Dynabeads (Invitrogen) coupled with specific antibody. Beads were then washed according to the Upstate protocol and DNA was de-crosslinked and eluted by overnight incubation in elution buffer at 65°C. Eluted DNA and an aliquot of the input DNA were treated with Proteinase K and RNase A (final concentrations of 125 µg/ml) at 55°C overnight. DNA was precipitated in ethanol after phenol/chloroform extraction and resuspended in Tris-EDTA (10/0.1) pH 7 buffer.
Label Cy3
Label protocol DNA samples were amplified using the WGA2 kit (Sigma Corp) according to the provided protocol, except the first fragmentation step was omitted. ChIP samples were amplified undiluted, whereas input control samples were amplified at a starting concentration of 1-2 ng/µl. After amplification, IP and input DNAs were labeled with Cy5 and Cy3 nonamers, respectively (TriLink Biotechnologies) using random primers and linear amplification with Klenow fragment of E. coli DNA Polymerase I (eBioscience Biotechnologies).
 
 
Hybridization protocol Arrays were hybridized in Maui hybridization stations following Nimblegen protocols (http://www.nimblegen.com/products/lit/lit.html).
Scan protocol Hybridized arrays were scanned on an Agilent Technologies DNA Micraarray Scanner model G2505B. Two-channel fluorescence intensities were extracted from the resulting TIFF files using NimbleScan v2.4 (Nbs1) or Nimblescan v2.6 (Pol2) software.
Description ChIP-chip wild-type splenic B cells Pol2
Data processing MA2C (Robust, C=2)
 
Submission date Oct 20, 2010
Last update date Jan 21, 2011
Contact name Richard E Baker
E-mail(s) richard.baker@umassmed.edu
Phone 508-856-6046
Organization name University of Massachusetts Medical School
Department Microbiology & Physiological Systems
Street address 55 Lake Avenue North
City Worcester
State/province MA
ZIP/Postal code 01655
Country USA
 
Platform ID GPL11075
Series (1)
GSE24827 Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig loci in activated B cells

Data table header descriptions
ID_REF
VALUE log2 (ChIP/Input) ratio, normalized

Data table
ID_REF VALUE
CHR01FS003000001 -0.382141
CHR01FS003000108 -0.993013
CHR01FS003000218 0.318322
CHR01FS003000303 -1.258897
CHR01FS003000403 -0.676291
CHR01FS003000523 0.565368
CHR01FS003000618 0.516454
CHR01FS003000772 -1.180932
CHR01FS003000852 0.011840
CHR01FS003000952 -0.294009
CHR01FS003001072 1.206928
CHR01FS003001172 1.156243
CHR01FS003001297 1.082539
CHR01FS003001377 -0.367059
CHR01FS003001483 -0.174994
CHR01FS003001587 0.420489
CHR01FS003001697 0.876931
CHR01FS003001787 -0.107705
CHR01FS003001877 0.532747
CHR01FS003002047 0.055263

Total number of rows: 2101986

Table truncated, full table size 54273 Kbytes.




Supplementary file Size Download File type/resource
GSM611074_01_Pol2_rep1_CY3.pair.gz 34.1 Mb (ftp)(http) PAIR
GSM611074_01_Pol2_rep1_CY5.pair.gz 33.9 Mb (ftp)(http) PAIR
Processed data included within Sample table

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