GSM9267467: BMDM KO, MafB2, Rep2; Mus musculus; OTHER
Bone marrow derived macrophages BMMos were isolated from BM using Monocyte Isolation Kit (BM) (Miltenyi Biotec, 130100629) and were seeded in tissue culture-treated plates and cultured for 4 days in RPMI supplemented with 10% FBS, 1 mM Sodium Pyruvate (Thermo Fisher Scientific, 11360070), 1X Minimum Essential Medium Non-Essential Amino Acids (MEM NEAA; Thermo Fisher Scientific, 11140050), 50 µM β-mercaptoethanol (Thermo Fisher Scientific, 31350010) and 40 ng/ml Csf-1 (Peprotech, 31502) to differentiate them into BMDMs.  Large peritoneal macrophages Lyz2CreMafbfl/fl mice and Mafbfl/fl littermate controls were terminally anesthetized and peritoneal lavage was obtained by injecting 10 ml PBS (Thermo Fisher Scientific, 14190094) containing 10 mM EDTA into the peritoneal cavity and collecting the washout. LPMs were FACS sorted from the peritoneal lavage as CD45+Lin−Ly6C−CD11b+CD115+F4/80+MHC-II− live cells.  Human monocyte-derived macrophages Buffy coats from healthy volunteers were obtained through the Belgian Red Cross. Peripheral blood mononuclear cells were isolated by density gradient centrifugation on Lymphoprep (1.077 g/ml; StemCell Technologies). Classical monocytes were negatively selected using the Classical Monocyte Isolation Kit (Miltenyi Biotec, 130117337) according to the manufacturer's instructions. Monocytes were seeded in Petri dishes (10 × 106 cells/dish) in RPMI supplemented with 10% FBS, 1× MEM NEAA (Thermo Fisher Scientific, 11140050), 50 µM β-mercaptoethanol, and 50 ng/ml recombinant human CSF-1 (Immunotools, 11343115). Fresh differentiation medium was added on day 3. On day 7, monocyte-derived macrophages (MDMs) were detached using cold PBS containing 0.25 mM EDTA. CUT&RUN was performed using the CUTANA ChIC/CUT&RUN Kit (EpiCypher, 141048) according to the manufacturer's instructions, with minor modifications. BMDMs or LPMs from Mafbfl/fl or Lyz2CreMafbfl/fl mice, as well as human MDMs from healthy donors, were washed with ice-cold PBS and fixed with 0.1% formaldehyde in PBS for 2 min at room temperature. Fixation was quenched with 0.125 M glycine (Merck, 104691000). For each CUT&RUN sample, 0.5 × 106 fixed cells were incubated with 0.5 µg antibody. The following antibodies were used: IgG (EpiCypher, 13-0042) MafB1 (Sigma, HPA005653) MafB2 (Cell Signaling Technology, 41019) MafB3 (Proteintech, 20189-1-AP) CUT&RUN libraries were prepared using the CUTANA CUT&RUN Library Prep Kit  (EpiCypher, 141001) following the manufacturer's instructions and sequenced on a NovaSeq 6000 (Illumina) with an S4 flow cell at a depth of 10 million reads per sample.