title: GSM2974744: NOD_BMDM_ATAC_notx_rep1; Mus musculus; ATAC-seq
library_construction_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.
attributes.tag: GEO Accession
attributes.value: GSM2974744