title: GSM8746337: Macro  Mafb KO, rep2, H3K27 antibody; Mus musculus; OTHER
library_construction_protocol: Bone marrow monocytes from Mafbfl/fl or Lyz2CreMafbfl/fl mice were isolated using Monocyte Isolation Kit (BM) (Miltenyi Biotec, 130100629) and were seeded in tissue culture-treated 6-well plates (Greiner, 657160) and cultured for 4 days in RPMI supplemented with 10% FBS, 1 mM Sodium Pyruvate (Gibco, 11360070), 1X MEM Non-Essential Amino Acids Solution (Gibco, 11140050), 50 µM β-mercaptoethanol (Gibco, 31350010) and 40 ng/mL CSF-1 (Peprotech, 315-02) to differentiate them into bone marrow monocyte derived macrophages (BMDM). H3K27Ac CUT&RUN was performed with a CUTANA ChIC/CUT&RUN Kit (EpiCypher, 141048) according to manufacturer's instructions, with modifications. BMDM from Mafbfl/fl or Lyz2CreMafbfl/fl mice were washed with ice-cold PBS and fixed with 0.1 % formaldehyde in PBS for 2 min at room temperature. Fixation was quenched by adding glycine (Merck, 104691000) to 0.125 M.  For each CUT&RUN sample, 1x 106 fixed cells and 0.5 µg antibodies were used. Anti-H3K27Ac (MA5-23516) and IgG Negative Control Antibody (EpiCypher, 13-0042) were used. CUT&RUN libraries were prepared using were prepared with a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, E7645L) according to manufacturer's instructions. These libraries were sequenced on an NovaSeq 6000 (Illumina) sequencer on an XP flow cell.