title: GSM2974655: C57_BMDM_Bisulfite; Mus musculus; Bisulfite-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. Bisulfite-seq: Bisulfite sequencing was performed as described in (Urich et al., 2015). DNA from C57Bl/6J and SPRET/EiJ BMDMs was isolated using the PureLink Genomic DNA Kit (Thermo Fisher Scientific) as described by the manufacturer. 2 μg of gDNA supplemented with 0.5% non-methylated ʎ- DNA (Promega) was used as input. DNA was fragmented to a 200 bp peak size using the Covaris microTube sonicator, size selected, end-repaired, adenylated and methylated adapters ligated exactly as detailed in Urich et al. Cytosine to uracil conversion was performed with the EZ DNA methylation-Gold kit (Zymo Research) with 450ng of adapter-ligated gDNA input as described within. Following bisulfite-treated DNA purification, reactions were amplified with four cycles, purified using one volume of AMPure XP bead solution and sequenced on an Illumina NextSeq 500 for 25 million and 31 million reads for C57Bl/6J and SPRET/EiJ, respectively. Conversion rates were 99.69%.
attributes.tag: GEO Accession
attributes.value: GSM2974655