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Sample GSM2974654

Query DataSets for GSM2974654

Status

Public on May 17, 2018

Title

C57_BMDM_ATAC_notx_rep2

Sample type

SRA

Source name

bone marrow derived macropages (BMDM)

Organism

Mus musculus

Characteristics

strain: C57BL/6J
tissue: bone marrow derived macropages (BMDM)
culture protocol: 7 day cultured BMDMs
ligands in culture: no treatment
chip antibody: NA

Extracted molecule

genomic DNA

Extraction protocol

Femur, tibia and iliac bones from the different mouse strains were flushed with DMEM high glucose (Corning) and red blood cells were lysed using red blood cell lysis buffer (eBioscience). After counting, 20 million bone marrow cells were seeded per 15cm non-tissue culture plates in DMEM high glucose (50%) with 20% FBS (Omega Biosciences), 30% L929-cell conditioned media (as source of M-CSF), 100 U/ml penicillin/streptomycin+L-glutamine (Gibco) and 2.5μg/ml Amphotericin B (HyClone). After 4 days of differentiation, 16.7 ng/ml mouse M-CSF (Shenandoah Biotechnology) was added to the media. After an additional 2 days of culture, non-adherent cells were washed off with room temperature DMEM high glucose and macrophages were obtained as a homogeneous population of adherent cells which were scraped and subsequently seeded onto tissue culture-treated petri dishes overnight in DMEM high glucose containing 10% FBS, 100 U/ml penicillin/streptomycin+L-glutamine, 2.5μg/ml Amphotericin B and 16.7 ng/ml M-CSF. For KLA activation, macrophages were treated with 10 ng/mL KLA (Avanti Polar Lipids) for 1 or 6 hours.
ATAC-seq: To approximately 150k nuclei in 22.5ul GRO freezing buffer, isolated as described for GRO-seq above, 25ul DNA Tagmentation buffer was added, reaction mixed and 2.5uL DNA Tagmentation Enzyme mix (Nextera DNA Library Preparation Kit, Illumina) added. Mixture was incubated at 37°C for 30 minutes and subsequently purified using the Zymogen ChIP DNA purification kit as described by the manufacturer. DNA was amplified using the Nextera Primer Ad1 and a unique Ad2.n barcoding primers using NEBNext High-Fidelity 2X PCR MM for 10 cycles. PCR reactions were purified using 1.5 volumes of SpeedBeads in 2.5M NaCl, 20% PEG8000, size selected using PAGE/TBE gels for 160 – 280 bp and DNA eluted as described for GRO-seq.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Description

ATAC-seq
Processed data file:
peaks_C57_ATAC_notx_opt.
Processed data file available in:
ATAC-seq_processed_data_files.tar

Data processing

Fastq files from sequencing experiments were mapped to the custom genome using default parameters for bowtie2 and shifted back to reference coordinates. HOMER was used for peak calling. IDR was applied to both replicates and the optimal peak file was used for further downstream analysis
genome build: mm10

Submission date

Jan 31, 2018

Last update date

Mar 11, 2021

Contact name

Christopher Glass

E-mail(s)

ckg@ucsd.edu

Phone

858-534-6011

Organization name

University of California, San Diego