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| Status |
Public on Sep 20, 2023 |
| Title |
Abl1_RRBS_med_5azadC_rep3 |
| Sample type |
SRA |
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| Source name |
Abl.1_2µM_5-aza-dC_day2_rep3
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| Organism |
Mus musculus |
| Characteristics |
strain: 129S1/SvImJ x CAST/EiJ F1
tissue: immortalised B cells
drug treatment: treated
library replicates: 3
Sex: female
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| Treatment protocol |
5-aza-2'-deoxycytidine (Sigma, A3656) was diluted in DMSO at a concentration of 10mM. Cells were grown in grown in growth media containing 0.2μM, 2μM and 10μM of 5-aza-2'-deoxycytidine. On Day 2, live cells were collected after sucrose gradient centrifugation (Histopaque®-1077, Sigma, Cat 10771), and genomic DNA was prepared for RRBS.
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| Growth protocol |
Immortalised B-cell llines were cultured in Roswell Park Memorial Institute medium (Gibco), containing 15% FBS (Sigma), 1X L-Glutamine (Gibco), 1X Penicillin/Streptomycin (Gibco) and 0.1% β-mercaptoethanol (Sigma).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
2x10^5 cells were spun down, washed using PBS and flash frozen on dry ice for DNA extractions for RRBS. Dead cells were removed from these samples by sucrose gradient centrifugation. (Histopaque®-1077, Sigma, Cat 10771), before sample preparation using conditions specified by the manufacturer.
Libraries were generated from 50ng input DNA using a scaled-down (half reactions) of the NuGEN Ovation RRBS Methyl-Seq System (Tecan) following the manufacturer's recommendation. Libraries were PCR amplified for 11 cycles.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Cell line Abl.1, in growth medium with 2µM 5-aza-dC, cells collected on Day 2, Library preparation replicate 3
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| Data processing |
Reference preparation: we created two custom parental genomes (“pseudogenomes” (Crowley et al., 2015; Zou et al., 2014); or “pseudocontigs” when we have phasing by contigs) for each organism, we used a custom script (all custom software tools mentioned are available on Github https://github.com/gimelbrantlab/QCumber). We also created a vcf-file with one allele considered as a reference (maternal 129S1 or first phased allele) and the other as an alternative allele. Only heterozygous sites were used.
RRBS reads were aligned to the mouse mm10 genome using BSmap3 with flags -v 0.05 -s 16 -w 100 -S 1 -p 8 –u.
Custom scripts written in Perl were used to calculate the methylation percentage for CpGs covered by 4 or more reads at locations of known SNPs between parental genomes. Briefly, VCF files containing SNPs between 129 and Cast strains were filtered to exclude calls that did not pass minimum requirements as well as C->T or G->A calls. For each SNP, RRBS reads that overlapped that SNP were extracted, and the methylation status of genomic cytosines was calculated by dividing the number of unconverted (methylated) cytosines (C) by the total number of unconverted (C) or converted (T) cytosines.
methylation status of all cytosines on reads overlapping a SNP were aggregated by SNP status to create a methylation average for the reference and alternate genotype.
Genome_build: mm10
Supplementary_files_format_and_content: bed files
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| Submission date |
Jan 21, 2020 |
| Last update date |
Sep 20, 2023 |
| Contact name |
Saumya Gupta |
| E-mail(s) |
saumya.sourire@gmail.com
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| Organization name |
Dana-Farber Cancer Institute
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| Department |
Cancer Biology
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| Street address |
450 Brookline Ave
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE144006 |
Mechanism of monoallelic expression and allelic rheostat role of DNA methylation (RRBS) |
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| Relations |
| BioSample |
SAMN13895984 |
| SRA |
SRX7586598 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4277990_Abl1_RRBS_med_5azadC_rep3.bed.gz |
13.1 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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