title: GSM7063455: Microglia, PLACseq, SALL1 Enhancer WT, Rep2; Mus musculus; OTHER
library_construction_protocol: Mouse brains were homogenized as previously described (Gosselin et al. 2014)  by gentle mechanical dissociation. Cells were then incubated in staining buffer on ice with anti-CD16/32 blocking antibody (BioLegend 101319) for 15 minutes, and then with anti-mouse anti-CD11b-APC (BioLegend 101212), anti-CD45-Alexa488 (BioLegend 103122), and anti-CX3CR1-PE (BioLegend 149006) for 25 minutes. Cell preparations for H3K27ac ChIP-seq were fixed with 1% formaldehyde for 10 minutes and quenched with 0.125M glycine for 5 minutes after staining, and subsequently washed three times. Cells were washed once and filtered through a 40 M cell strainer. Sorting was performed on a Sony MA900 or MoFlo Astrios EQ cell sorter. Microglia were defined as events that were DAPI negative, singlets, and CD11b+CD45lowCX3CR1+. Sorted microglia were pelleted at 800xg for 5 minutes, pellets were then snap frozen and stored at -80°C prior to library preparation. Brain nuclei were isolated as previously described (Nott et al., 2019), with initial homogenization performed with either 1% formaldehyde in Dulbecco's phosphate buffered saline or 2mM DSG (Proteochem) in Dulbecco's phosphate buffered saline. Nuclei were stained overnight with PU.1-PE (Cell Signaling 81886S), OLIG2-AF488 (Abcam 225099) or SALL1 AF647 (Thermo, clone NRNSTNX 51-9279-82) or NEUN-AF488 (Millipore MAB 377X). Nuclei were washed the following day with 4 mL FACs buffer, passed through a 40 uM strainer, and stained with 0.5 ug/mL DAPI. Nuclei for each cell type were sorted with a Beckman Coulter MoFlo Astrio EQ cell sorter and pelleted at 1600xg for 5 minutes at 4°C in FACs buffer. Nuclei pellets were snap frozen and stored at -80°C prior to library preparation. PLAC-seq libraries were prepared for ex vivo microglia as previously described with minor modifications (Nott et al., 2019; Fang et al., 2016). In situ Hi-C was performed as described previously, with minor modifications (Heinz et al., 2018).