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| Status |
Public on Aug 04, 2015 |
| Title |
RAG2ko_thy_mm_TCRBetaTg_RNAseq C21 |
| Sample type |
SRA |
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| Source name |
thymocytes
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| Organism |
Mus musculus |
| Characteristics |
cell type: thymocytes
genotype: Rag2: -/-; TCRbeta: +/+/tg
strain: C57BL/6
age: 5
Sex: female
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Thymocytes were crosslinked for 15 minutes with 1% formaldehyde, and the crosslinking reaction was quenched with 0.125 M glycine. Cells were washed twice with ice-cold PBS containing 1 mM PMSF and 1 µg/mL pepstatin A. Five million crosslinked cells were resuspended in MNase digestion buffer (50 mM Tris-HCl, pH 7.6; 1 mM CaCl2, 0.2% Triton X, 5 mM sodium butyrate, 1 mM PMSF, 1 µg/mL pepstatin A). Micrococcal nuclease (MNase, 0.2 U) was added to the sample, which was incubated at 25 ºC for 5 minutes. The nuclease digestion was terminated with 5 mM EDTA. Crosslinks were reversed using an overnight incubation at 65 ºC in the presence of 0.5% SDS and 200 µg/mL Proteinase K. DNA was isolated by standard phenol:chloroform extraction and ethanol precipitation. The sample was then subjected to RNAse digestion (10 µg/mL RNAse A at 37 ºC for 1 hour) and a second proteolysis step to remove the RNAse (200 µg/mL Proteinase K, 0.3% SDS, 65 ºC, 1 hour). Again, the DNA was isolated by phenol:chloroform extraction and ethanol precipitation and resuspended in water. The sample was run on a 2% agarose gel, and the ~150 bp mononucleosome band was excised and purified using the Qiagen Gel Extraction kit.
libraries were prepared and sequenced following standard Illumina protocols
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
C21
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| Data processing |
Libraries were run on Illumina HiSeq Sequencers and Illumina tools were used to generate fastq files from RTA output.
the short reads were then aligned to the mm9 genome with Bowtie (versions 0.12.8 to 1.0.0) (Langmead et al., 2009).
After sorting the resulting alignment files, custom software was used to determine mean density of reads around RefSeq promoters that overlapped a Rag2 peak.
MNase reads were shifted by half a nucleosome size towards the center of the nucleosome.
The resulting data was imported into R (version 3.1) for visualization.
Genome_build: GRCm37/mm9
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| Submission date |
Jun 02, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Yaakov Maman |
| Organization name |
Bar-Ilan University
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| Department |
Azrieli Faculty of Medicine
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| Lab |
Genome Instability & Cancer
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| Street address |
Henrietta Szold 8
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| City |
Safed |
| ZIP/Postal code |
1311502 |
| Country |
Israel |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE69474 |
RAG Represents a Widespread Threat to the Lymphocyte Genome - (Mnase-seq) |
| GSE69478 |
RAG Represents a Widespread Threat to the Lymphocyte Genome |
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| Relations |
| BioSample |
SAMN03757532 |
| SRA |
SRX1046526 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1701776_C21.bw |
71.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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