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Sample GSM1467675

Query DataSets for GSM1467675

Status

Public on Jul 18, 2017

Title

RRBS_mESC_RA_p53_KO_1

Sample type

SRA

Source name

RRBS_mESC_RA_p53_KO

Organism

Mus musculus

Characteristics

age: E2.5
genotype/variation: p53-/-
tissue/cell type: mouse embryonic stem cells (mESC)
cell state: induced to differentiate by RA

Extracted molecule

genomic DNA

Extraction protocol

129s-Trp53tm2Tyj/J p53+/- (Jackson Laboratory, Mt Desert Is., ME, USA) heterozygous p53+/- male and female mice were time mated. E2.5 morulas were isolated from the uteri of female mice and allowed to attach to irradiated MEF feeder layers in 2i medium. Subsequently, 2i medium was changed daily. For differentiation experiments, feeders were depleted by 30–45 minutes of incubation on 0.1% gelatin, followed by gentle aspiration of purified mESCs. For RA-induced differentiation, feeder-depleted cells were seeded on gelatin and transferred to differentiation medium containing for 16 hours followed 1μM retinoic acid (RA). RA was replenished daily for 4 days.
Reduced representation bisulfite sequencing (RRBS) was performed according to a previously published protocol (Boyle, P. et al. 2012).
Briefly, genomic DNA was isolated using the PureLink genomic DNA kit (Invitrogen) according to the manufacturer’s instructions and subjected to enzymatic digestion by MSPI (New England Biolabs) for 16 hours. Klenow polymerase (New England Biolabs) was then added to each sample for end filling and poly-A tailing, followed by Truseq adapter ligation (Illumina). The library was then pooled and subjected to either bisulfite treatment as in the RRBS protocol or to an OX-RRBS protocol using the CEGX kit (Cambridge Epigentix). Validation of oxidation efficiency was done with the provided DNA sequences that were also A tailed and adapter ligated.

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

Reduced Representation

Instrument model

Illumina HiSeq 2000

Description

processed data file: SingleCpG_mESC_RRBS_oxRRBS.txt.gz

Data processing

The final RRBS and OX-RRBS libraries were sequenced using the Illumina Hiseq2500 to obtain 100 bp paired-end sequencing reads. Adapter trimming and quality filtering was performed with the trim_galore software using default parameters for RRBS analysis. BSMAP was used for read alignment (using genome build mm9) and extraction of single base-resolution methylation levels. Integration of biological replicates, 200 bp tiles and DMRs were calculated with the MethylKit package using a minimum coverage of 10, methylation difference of 30%, and q-value <= 0.01. Total methylation levels (5mC+5hmC) of single CpGs and 200bp tiles were obtained from the RRBS data. 5mC levels were obtained accordingly from the OX-RRBS data, and 5hmC levels were calculated as (Total - 5mC) levels. Only CpGs that were covered by all biological replicates and that had 5hmC>=0 values were used for the analysis
Genome_build: mm9
Supplementary_files_format_and_content:
'200bpTiles_Unified.txt.gz' containing genomic location tile, Total, 5hmc, 5hmc values of all conditions
'SingleCpG_mESC_RRBS_oxRRBS.txt.gz' containing genomic location CpG, Total and 5mc values of all mESC conditions

Submission date

Aug 07, 2014

Last update date

May 15, 2019

Contact name

Moshe Oren

Organization name

The Weizmann Institute of Science

Street address

234 Herzl St.

City

Rehovot