title: GSM5848609: Mammary gland macrophages replicate 2; Mus musculus; Bisulfite-Seq
library_construction_protocol: Normal mammary fat pad: Tissue was minced using scissors and placed and disrupted for 25 min at 37oC in a gentleMACS C tube using gentleMACS dissociator, in the presence of enzymatic digestion solution containing 1 mg ml-1 collagenase II, 1 mg ml-1collagenase IV and 70 unit ml-1 DNase. Primary tumor: Tissue was minced using scissors and treated with enzymatic digestion solution containing 3 mg ml-1 collagenase A and 70 unit ml-1 DNase in RPMI 1640 for 20 min at 37oC, with pipetting every 3 min. Foe BMDM extraction the Tibia and Femur leg bones were dissected Bones were flushed with MACS buffer using a 5 ml syringe with a 25 G needle. The flushed cells from the bone marrow were centrifuged at 500 g for 5 min and resuspended in red blood cell lysis buffer  cells were washed with PBS and resuspended in macs buffer. Post bisulfite adaptor tagging protocol by Clark et al 2017: WGBS libraries from low input material were prepared using a PBAT protocol initially developed for single-cell bisulfite sequencing. As input, 6 ng of purified DNA or 1,000 cells, which were lysed in RLT Plus buffer (Qiagen), were used. A total of 10 µl of cell suspension was applied to the protocol, including the following changes: A single preamplification step was performed for 90 min at 37°C. Fourteen cycles of library amplification were conducted with default PCR parameters. Libraries were purified by applying twice a 0.7x SPRI selection using AMPure XP beads (Agencourt, Indianapolis, IN, USA). The libraries' molarities were calculated based on concentrations measured with the Qubit dsDNA Assay (Thermo Fisher Scientific) and fragment size distribution measured with the high sensitivity D5000 Assay for the Agilent Tapestation (Agilent Technologies Germany GmbH & Co. KG, Waldbronn, Germany). Libraries were sequenced one lane per sample on a HiSeq X sequencer using the 150 base pairs (bp), paired-end mode (Illumina, San Diego, CA, USA) at the DKFZ Genomics and Proteomics Core Facility.