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Sample GSM3375721

Query DataSets for GSM3375721

Status

Public on Nov 02, 2018

Title

D2018A input

Sample type

SRA

Source name

embryonic stem cells

Organism

Mus musculus

Characteristics

genotype: TET1 mutation, D2018A
cell line: LF2
cell type: ESC
flag tag: C-terminal FLAG tag on TET1

Growth protocol

embryonic stem cells were passaged in LIF containing embryonic stem cells media (10% FBS)

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA was extracted with Qiagen Dneasy Blood & Tissue kit according to the manufacturer's protocol.
100 ng genomic DNA extracted from ES cells were fragmented in 50 μL Tagmentation buffer at 55°C. Fragmented DNA was purified by Zymo DNA clean&concentrator Kit (Zymo Research, Tustin, CA). Then, the selective 5hmC chemical labeling was performed in 25 μL glucosylation buffer (50 mM HEPES buffer pH 8.0, 25 mM MgCl2) containing above fragmented DNA, 100 μM N3-UDP-Glc, 1 μM β-GT, and incubated at 37°C for 2 hr. After purified in 45 μL ddH2O, 1.5 μL DBCO-PEG4-Biotin (Click Chemistry Tools, 4.5 mM stored in DMSO) was added and incubated at 37°C for 2 hr. The biotin labeled DNA was pulled down by 5 µL C1 Streptavidin beads (Life Technologies, Carlsbad, CA) for 15 min at room temperature. Next, the captured DNA fragments were subjected to 13 cycles of PCR amplification using Nextera DNA sample preparation kit (Illumina, San Diego, CA). The resulting amplified product was purified by 1.0X AMPure XP beads. Input library was made by direct PCR from fragmented DNA without chemical labeling and capture. The libraries were quantified by a Qubit fluorometer (Life Technologies) and sequenced on an Illumina HiSEQ2500 sequencer with paired-end 40-bp reads.

Library strategy

MeDIP-Seq

Library source

genomic

Library selection

5-methylcytidine antibody

Instrument model

Illumina HiSeq 2000

Description

DA_peaks.bed.gz

Data processing

Adaptors and low quality nucleotides were removed from raw sequencing paired-end reads by Trim_Galore.
Bowtie was used to map clean reads to mm9 reference genome.
Multi-mapped reads and duplicated reads were further removed using samtools.
Peak calling was performed by using MACS1.4
Genome_build: mm9
Supplementary_files_format_and_content: bed format files for peaks called in WT and DA samples.

Submission date

Sep 05, 2018

Last update date

Nov 02, 2018

Contact name

Xiaolong Cui

E-mail(s)

xiaolong.cui@northwestern.edu

Organization name

Northwestern University

Department

Department of Preventive Medicine