title: GSM8217825: H3K27AC_MONOCYTES_STROKE_2; Mus musculus; OTHER
library_construction_protocol: Bone marrows (from femurs) were homogenized and filtered through 40 μm cell strainers to obtain single cell suspensions. BM monocytes(CD11b+Ly6C+Ly6G-)andthelineage-(CD3, CD4, CD8a, CD19,Ter119)and Ly6G)negative myeloid fraction of BM (HSPC-enriched population)weresorted using aFACS Aria II cell sorter (BD Biosciences, Inc.). nuclei were extracted from 1·10^5 cells, washed twice and incubated with 10 μL of activated concanavalin A beads (ThermoFisher) for 10 min atRT. The beads were thenresuspended in cold antibody buffer containing primary antibodies(against H3K27ac, H3K4me1 or H3K4me3)and the mixture was incubated overnight at 4°C on anutator. Then, the beads were washed twice and resuspended in secondaryH+Lantibody(NovusBio)dilutedin 50 μL digitonin buffer, followed by incubation for 30 min atRTon a rotator. Samples were then incubated with 2.5 µL CUTANA pAG-Tn5 (EpiCypher) for 1 h at RT on a nutator. DNA was tagmented for 1 hat 37°C using tagmentation buffer and tagmented chromatin fragments were released for 1 h at 58°C. 13 ul of tagged DNA was used to prepare the library using NEBNext® High-Fidelity 2X PCR Master Mix(NEB)with CUT&Tag specific PCR cycling parameters. DNA was purified using AMPureXP Beads (Beckman Coulter). The quality of the CUT&Tag libraries was assessed using the Agilent 2100 Bioanalyzer System (Agilent).