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Sample GSM5468456

Query DataSets for GSM5468456

Status

Public on Jul 24, 2021

Title

v65-WT_L1mdA

Sample type

SRA

Source name

V65 mESC

Organism

Mus musculus

Characteristics

cell type: mESC
strain: V65
genotype: WT

Treatment protocol

N/A

Growth protocol

mES cells were cultured in Knock Out DMEM media (Gibco) containing 15% FBS, 1% PEN/STREP, 1% glutamine, 1% NEAA, and 105 U LIF. For ES cell maintenance, dishes were coated with 0.2% gelatin and irradiated CD1 mouse embryonic fibroblasts (MEFs) were plated as a confluent layer of feeder cells. mES cells were seeded at a density of 50,000 cells per well of a six-well plate and were split every 3–4 days.

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA was isolated from cells through phenol-chloroform extraction
200 ng of genomic DNA was digested with 10 U of BsaWI (NEB), DdeI (NEB), Eco47I (Thermo Fisher Sc.) or MspI (Thermo Fisher Sc.), incubated for 16 h at 60°C (BsaWI) or 37°C (DdeI, Eco47I and MspI) and afterwards heat inactivated. 50 pmol of endonuclease specific hairpin-linker was ligated in a 20 µl reaction at 16 °C for 3 h. Bisulfite conversion was done using Zymo’s EZ DNA Methylation-Gold Kit according to manufacturer’s instructions. Bisulfite treated Hairpin-DNA was eluted in 20 µl Elution Buffer. PCR was performed in 30 µl reactions with 2 µl of Hairpin-Bisulfite template and 37 PCR cycles using HotFirePol®DNA Polymerase (Solis BioDyne) or HotStarTaq DNA Polymerase (Qiagen). PCR products were purified from a 1.2% agarose gel using Avegene’s GenepHlow™ Gel/PCR Kit according to manufacturer’s instructions and eluted in 20 µl 0.1x TE buffer. Amplicons were indexed in a 50 µl PCR using specific TruSeq indices and 6 PCR cycles. Index PCRs were purified with 1.1x AMPure XP beads (Beckman Coulter). Amplicons were sequenced on an Illumina MiSeq in a 2x250 bp sequencing mode

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina MiSeq

Description

BS_hairpin.csv

Data processing

Library strategy: BS-Amplicon
CpG methylation states were determined using BiQAnalyzerHT, filtering for a sequence identity of >= 0.8 (reference sequence for IAP and IAPez have been shortened in order to obtain a better mapping of the sequencing reads) and strand-specific methylation patterns were obtained using Hairpinanalyzer V0.3 (Mathias Bader and Julia Arand, available under: https://github.com/PascalGiehr/Hairpinanalyzer), filtering for a linker conversion rate of >= 0.8. H(O)TA was applied in order to determine enzyme efficiencies.
Genome_build: mm9
processed data files format and content: csv containing read count statistics

Submission date

Jul 23, 2021

Last update date

Jul 25, 2021

Contact name

Helene Kretzmer

E-mail(s)

kretzmer@molgen.mpg.de

Organization name

Max Planck Institute for Molecular Genetics

Department

Genome Regulation