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Sample GSM2946100

Query DataSets for GSM2946100

Status

Public on May 02, 2018

Title

Tet1_WT_ChIP-seq_R26_1

Sample type

SRA

Source name

TS-Rs26

Organism

Mus musculus

Characteristics

cell line: TS-Rs26
cell type: Mouse trophoblast stem cells
genotype: WT

Growth protocol

TS-Rs26 trophoblast stem cells were routinely cultured in complete TS medium, consisting of 30% TS base medium (RPMI 1640 w/ glutamine (invitrogen 21875-0910) + 20% FBS + Na-pyruvate + 2ME + antibiotics) and 70% MEF conditioned TS base + bFGF + Heparin. Differentiation was induced by withdrawal of bFGF, heparin and conditioned medium.

Extracted molecule

genomic DNA

Extraction protocol

Total RNA was extracted using TRI reagent (Sigma T9424), followed by DNase treatment using the TURBO-DNA-free kit (Life Technologies AM1907). For ChIP-seq, cells (1-2x108) were fixed in 2 mM DSG (Sigma 80424) for 45min and 1% formaldehyde in TS base media for 12 min, both at room temperature (RT). Fixation was stopped by 0.125M glycine. Cells were washed in buffer 1 (10 mM Hepes pH 7. 5, 10 mM EDTA, 0.5 mM EGTA, 0.75% Triton X-100), buffer 2 (10 mM Hepes pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and lysed in the lysis/sonication buffer (150 mM NaCl, 25 mM Tris pH 7.5, 5 mM EDTA, 0.1% Triton, 1% SDS, 0.5% sodium deoxycholate on ice for 30min. Chromatin was sonicated 30sec on/30off for 25-30 cycles using the BioRuptor (Diagenode), diluted 1/10 with the dilution buffer (150 mM NaCl, 25 mM Tris pH 7.5, 5 mM EDTA, 1% Triton-X 100, 0.1% SDS, 0.5% sodium deoxycholate) and pre-cleared with protein G magnetic Dynabeads (Invitrogen 10004D) pre-blocked with BSA and tRNA. Immunoprecipitation was performed with 350 μg chromatin and 10 μg of antibody over night at 4oC and pre- blocked magnetic beads were added next morning for 7-8h. Beads were washed three times each with buffer A (150 mM NaCl, 25 mM Tris pH 7.5, 5 mM EDTA, 1% Triton-X 100, 0.1% SDS, 0.5% Sodium Deoxycolate), buffer B (50 mM Tris pH8.0, 500 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, 1 mM EDTA), buffer C (50 mM Tris pH8.0, 250 mM LiCl, 0.5% sodium deoxycholate, 1% NP40, 1 mM EDTA). DNA was eluted (1%SDS, 0.1M NaHCO3), reverse cross-linked overnight at 65oC and purified on PCR purification columns (Qiagen). For BS-seq, DNA from vector control (WT) and Tet1 KO cells was bisulfite-converted using the EpiTect kit (Qiagen). The Cdh1 locus was amplified with KAPA HiFi Hotstart Uracil+ ReadyMix (Roche Diagnostics Ltd KK2801).
To generate RNA libraries, mRNA was isolated using the Dynabeads mRNA purification kit (Life Technologies 61006) and processed into indexed, strand-specific libraries using the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre SSV21106). Libraries were quantified and assessed using the KAPA Library Quantification Kit (KAPA Biosystems KK4824) and Bioanalyzer 2100 system (Agilent). Indexed libraries were sequenced with a 100bp single-end protocol on an Illumina HiSeq2500 sequencer. ChIP sequencing libraries were quantified as above by Bioanalyzer 2100 system (Agilent) and using the KAPA Library Quantification Kit (KAPA Biosystems KK4824). Indexed libraries from ChIP and Input samples were sequenced with a 50bp paired-end protocol on an Illumina HiSeq2500 sequencer. For BS-seq, libraries were amplified and indexed using KAPA HiFi HotStart ReadyMix (Roche Diagnostic Ltd KK2602). Libraries were quantified as above by Bioanalyzer 2100 system (Agilent) and using the KAPA Library Quantification Kit (KAPA Biosystems KK4824, and sequenced with a 150bp paired-end protocol on a MiSeq sequencer (Illumina), with 10% PhIX spike-in.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

lane5037_ATCACGTT_R26_1
Chromatin immunoprecipitation (ChIP) and sequencing library generation was carried out as described previously (Latos et al., 2015b). 150mg chromatin and 5mg Tet1 antibody (GeneTex GTX125888) were used for each ChIP. ChIP as well as Input libraries were generated in biological triplicates. Sequencing libraries were quantified as above by Bioanalyzer 2100 system (Agilent) and using the KAPA Library Quantification Kit (KAPA Biosystems KK4824).

Data processing

Reads trimmed with Trim Galore using standard protocols.
RNA-Seq reads mapped to GRCm38/mm10 using hisat
RNA-Seq cpm generated by edgeR
ChIP-Seq reads mapped to GRCm38/mm10 using Bowtie
ChIP-Seq peaks called using MACS2 and standard parameters
Bisulfite sequencing data was mapped to the Mus musculus GRCm38 genome assembly and processed using Bismark to find the percent methylation for each CpG
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: bed file contains MACS2 peak calls for Tet1 WT; csv files contain normalised cpm data per gene for each sample generated using edgeR; cov files are tab delimited files containing CpG coverage per genomic region.

Submission date

Jan 24, 2018

Last update date

May 02, 2018

Contact name

Myriam Hemberger

Organization name

Babraham Institute

Department

Epigenetics

Street address

Babraham Hall