|
| Status |
Public on Jan 31, 2016 |
| Title |
Pro-B.1 mono |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
mononucleosomal DNA from Rag2-/- pro-B Abelson-transformed cell line
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype/variation: Rag2-/-
cell type: pro-B Abelson-transformed cell line
fraction: mononucleosomal DNA
|
| Growth protocol |
Rag2-/- pro-B Abelson-transformed cell line was grown in RPMI + 10% FBS + P/S + B-Me
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were grown exponentially until they reached confluence. Cells were crosslinked with formaldheyde for 10' at RT and reaction was stopped with 0.125 M of glycine for 5’ at RT. Cells were then washed twice with cold PBS and nuclei were excracted using a sucrose buffer [0.3 M sucrose, 2 mM MgOAc2, 1% Triton X-100, 10 mM HEPES (pH 7.8)]. MNase digestion was performed at 37°C for 5' in MNase digestion buffer [25mM KCl, 4 mM MgCl2, 1 mM CaCl2, 12.5% glycerol and 50 mM TrisHCl (pH 7.4)] and reaction was terminated using an equal amount of MNase stop buffer [2% SDS, 0.2 M NaCl, 20 mM EGTA, 20 mM EDTA and 50 mM TrisHCl (pH 8)]. Samples were RNased for 1hr at 37ºC and decrosslinked O/N at 65ºC together with proteinase K. Digests were extracted with phenol:chloroform, and DNA recovered by isopropanol precipitation. DNA was loaded on a prestained 2% TAE agarose gel and mononucleosomal DNA exctracted.
|
| Label |
Cy3
|
| Label protocol |
0.5 µg of either mononucleosomal DNA or bare genomic DNA were labeled using the NimbleGen Dual-Color DNA labeling Kit (REF 06370250001) and following manufacturer's protocol for CGX arrays.
|
| |
|
| Channel 2 |
| Source name |
MNased digested bare genomic DNA from Rag2-/- pro-B Abelson-transformed cell line
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype/variation: Rag2-/-
cell type: pro-B Abelson-transformed cell line
fraction: MNased digested bare genomic DNA
|
| Growth protocol |
Rag2-/- pro-B Abelson-transformed cell line was grown in RPMI + 10% FBS + P/S + B-Me
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were grown exponentially until they reached confluence. Cells were crosslinked with formaldheyde for 10' at RT and reaction was stopped with 0.125 M of glycine for 5’ at RT. Cells were then washed twice with cold PBS and nuclei were excracted using a sucrose buffer [0.3 M sucrose, 2 mM MgOAc2, 1% Triton X-100, 10 mM HEPES (pH 7.8)]. MNase digestion was performed at 37°C for 5' in MNase digestion buffer [25mM KCl, 4 mM MgCl2, 1 mM CaCl2, 12.5% glycerol and 50 mM TrisHCl (pH 7.4)] and reaction was terminated using an equal amount of MNase stop buffer [2% SDS, 0.2 M NaCl, 20 mM EGTA, 20 mM EDTA and 50 mM TrisHCl (pH 8)]. Samples were RNased for 1hr at 37ºC and decrosslinked O/N at 65ºC together with proteinase K. Digests were extracted with phenol:chloroform, and DNA recovered by isopropanol precipitation. DNA was loaded on a prestained 2% TAE agarose gel and mononucleosomal DNA exctracted.
|
| Label |
Cy5
|
| Label protocol |
0.5 µg of either mononucleosomal DNA or bare genomic DNA were labeled using the NimbleGen Dual-Color DNA labeling Kit (REF 06370250001) and following manufacturer's protocol for CGX arrays.
|
| |
|
| |
| Hybridization protocol |
34 µg of test sample and 34 µg of reference sample were combined and resuspended in 12.3 µl of water. Hybridization buffer was added and hybridization permormed frollowing the CGX NimbleGen protocol.
|
| Scan protocol |
Arrays were scanned on an GenePix 4000B Microarray Scanner following manufacturer's protocol instructions.
|
| Description |
ProB1
|
| Data processing |
Arrays were processed using Nimblegen's standard protocol for data extraction.
|
| |
|
| Submission date |
Nov 15, 2015 |
| Last update date |
Jan 31, 2016 |
| Contact name |
Guray Kuzu |
| Organization name |
Massachusetts General Hospital
|
| Street address |
185 Cambridge Street
|
| City |
Boston |
| ZIP/Postal code |
02114 |
| Country |
USA |
| |
|
| Platform ID |
GPL21139 |
| Series (1) |
| GSE75018 |
Large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination at the IgH locus in a cell-type specific manner |
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