|
| Status |
Public on Jan 21, 2011 |
| Title |
03_Pol2_rep2 |
| 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 22, 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 |
GPL11091 |
| Series (1) |
| GSE24827 |
Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig loci in activated B cells |
|
