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Sample GSM2300994

Query DataSets for GSM2300994

Status

Public on Sep 02, 2016

Title

B6_thymocytes_input

Sample type

SRA

Source name

Thymocytes

Organism

Mus musculus

Characteristics

tissue: Thymus

Extracted molecule

genomic DNA

Extraction protocol

Thymocytes (1.5 × 10^8 cells) from 4 to 5-weeks-old C57BL/6, Zfatf/f or Zfatf/f-LckCre mice were crosslinked with 1% formaldehyde for 10 min at room temperature. Crosslinking reaction was quenched with 125 mM glycine for 5 min. The cells were rinsed with PBS twice and incubated in 600 µl of MNase treated buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% NP-40, 5 mM CaCl2) containing 375 unit MNase (Roche) for 0.5 h at 37°C. The MNase reaction was stopped by adding 600 µl of lysis buffer (100 mM Tris-HCl, pH 8.0, 20 mM EDTA, 2% SDS, protease inhibitor cocktail (Roche)), and the lysate was sonicated using a Bioruptor (Cosmo Bio, Tokyo, Japan) for two cycles of one minute with 30 seconds on/off. The sonicated lysate was subjected to immunoprecipitation with a monoclonal antibody against ZFAT (prepared in Dr. Shirasawa's lab), H3K9me3 (MBL), or H3K9ac/K27ac (MBL).
The immunoprecipitated DNA was purified using MinElute Reaction Cleanup kit (Qiagen) according to the manufacturer’s instructions. ChIP-seq libraries were prepared using NEBNext ChIP-seq Library Prep Master Mix Set and Multiplex Oligos for Illumina (New England Biolabs) according to manufacturer's instructions. ChIP or input DNA (0.1 ~ 1 ng) was subjected to end repair, dA-tailing, and adaptor-ligation, and amplified by 6-13 cycles of PCR.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 1500

Data processing

Paired-end sequencing (101bp x2) was performed on the HiSeq1500 system (illumina).
Basecalls were performed using the Real-Time Analysis (RTA) version 1.12.4.2 and CASAVA_v1.8.1.
Reads from each sample were first trimmed by removing adapters and low quality bases at ends using Trimmomatic 0.22.
Approximately the same number of reads (30 ~ 60 million reads) for a pair of ChIP and corresponding input libraries were aligned to the mouse reference genome (mm9) using the Burrows-Wheeler Aligner 0.6.2.
Uniquely mapped reads were selected by a custom script, converted from sam to bam using SAMtools 0.1.18. Reads with mapping quality < 20 were removed using SAMtools 0.1.18.
The resultant bam files were visualized using the Integrative Genomics Viewer (IGV). The uniquely mapped reads were analysed using the MACS algorithm implemented in the Avadis NGS softwere (Agilent) to identify ChIPseq peaks using the matched input DNA reads as a control.
Genome_build: mm9
Supplementary_files_format_and_content: MACS peak calling output

Submission date

Sep 01, 2016

Last update date

May 15, 2019

Contact name

Kazuhiko Nakabayashi

E-mail(s)

nakabaya-k@ncchd.go.jp

Organization name

National Research Institute for Child Health and Development

Department

Department of Maternal-Fetal Biology