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Sample GSM1215603

Query DataSets for GSM1215603

Status

Public on Jan 16, 2014

Title

Anti-CMS_5hmC_IP_Tet2kd_rep2

Sample type

SRA

Source name

Tet2kd mES cells

Organism

Mus musculus

Characteristics

cell line: V6.5
knockdown: Tet2 kd
dna enrichment: CMS antisera
biological replicate: 1

Treatment protocol

Cells stably depleted of Tet1 and Tet2 were obtained by electroporating V6.5 mESC with pSUPER-puro-Tet1shRNA or pSUPER-puro-Tet2shRNA (320V, 250F). Cells were selected by 1.5 g/ml puromycin for 7-10 days on puromycin-resistant mitomycine C-inactivated SNL76/7-4 feeder cells. Individual clones were picked and propagated in the absence of puromycin if the passage number was less than 10, or maintained in 1 g/ml puromycin if the passage number was longer than 10. Tet1 and Tet2 mRNA expression in individual clones was evaluated by qPCR.

Growth protocol

Mouse embryonic stem cells were cultured in Knockout D-MEM with 15% ES-qualified FBS (Gemini Bio-products), 2 mM L-glutamine with 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 50 units/ml penicillin/streptomycin and 1000 U/ml ESGRO® (LIF; Chemicon).

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA was sheared using Covaris Adaptive Focused Acoustic to the center at 150 bp. DNA fragments were size selected and ligated with methylated adaptors and treated with sodium bisulfite (invitrogen). The extra adapters were removed by AmpurXP (Beckman). The DNA was then denatured for 10 minutes at 95 °C (0.4 M NaOH, 10 mM EDTA), neutralized by the addition of cold 2M ammonium acetate (pH 7.0) for 10 min on ice. Then denatured DNA fragments were incubated with 1 μl anti-CMS antiserum in 1x IP buffer (10 mM sodium phosphate pH 7.0, 140 mM NaCl, 0.05% Triton X-100) for 2 h at 4 °C, and then precipitated with protein G beads. Precipitated DNA was eluted with proteinase K, purified with phenol chloroform, amplified and enriched by 4-6 cycles PCR using Pfu TurboCx hotstart DNA polymerase (Stratagene). DNA sequencing was carried out using Illumina Genome Analyzer 2 and HiSeq sequencing systems.

Library strategy

MeDIP-Seq

Library source

genomic

Library selection

5-methylcytidine antibody

Instrument model

Illumina HiSeq 1000

Data processing

Anti-CMS and Input reads derived from parental v6.5 mESC, Tet1 kd mESC, and Tet2 kd mESC input and CMS-IP were obtained and mapped against mm9 as previously described [Pastor et al. Nature 2011, PMID 21552279] by discarding all reads that map to multiple positions in the genome. Reads that map to the same genomic position were replaced by one representative. Mapping results from replicates were pooled.
In order to identify genome wide differentially hydroxymethylated regions (DHMRs) in parental v6.5 compared to Tet1, or Tet2 kd, respectively, we calculated library size normalized differential coverage of the CMS-IP data at genome wide 300 bp windows by employing a developmental version (v1.9.10) of the Bioconductor package MEDIPS (extend=200, uniq=F, window_size=300, BSgenome=BSgenome.Mmusculus.UCSC.mm9, diff.method = edgeR, p.vale = 1e-5), and adjacent significant windows with the same direction of 5hmC change were merged.
Genome_build: mm9
Supplementary_files_format_and_content: Result tables contain counts, rpkm, log2FC, and p-values at genome wide 300 bp windows as returned by the MEDIPS software for i) Tet1 kd mESC vs. v6.5mESC and ii) Tet2 kd mESC vs. V6.5mESC

Submission date

Aug 26, 2013

Last update date

May 15, 2019

Contact name

Lukas Chavez

E-mail(s)

l.chavez@dkfz.de

Organization name

German Cancer Research Center (DKFZ)

Street address

Im Neuenheimer Feld 280