GSM7081687: iMG, rep5; Homo sapiens; ATAC-seq
For iMGs, cells were carefully manually scraped from the Matrigel coated plate and were concentrated by a 300 rcf x 5 minutes. For oMGs, organoids were carefully collected, allowed to settle by gravity, and then dissociated mechanically in staining buffer (HBSS 1x with 1mM EDTA and 1% BSA) using a 2 ml polytetrafluoroethylene pestle. iMGs and oMGs were stained with the following Biolegend antibodies/dyes: CD64-APC (305014), CX3CR1 PCP-Cy5.5 (341614), CD14-488 (325610), CD11b-PE (301309), HLADR PE-Cy7 (307616), CD45-APC-CY7 (368516), and Zombie Violet (1:1000, Biolegend) for live/dead discrimination. xMGs were isolated and sorted at indicated time points following xenotransplantation. Briefly, mouse brains were homogenized as previously described by gentle mechanical dissociation (Gosselin et al., 2014) and stained with DAPI, CX3CR1-APC (Biolegend 149008), anti-human CD11b-PE (Biolegend 301306), anti-human CD45-FITC (Biolegend 368508). Microglia were purified with either a BD Influx  or FACS  AriaFusion. Approximately 30,000-50,000 isolated microglia were lysed in 50 µl lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL, CA-630, in water). Resulting nuclei were centrifuged at 500 rcf for 10 minutes. Pelleted nuclei were resuspended in 50 µl transposase reaction mix (1x Tagment DNA buffer (Illumina 15027866), 2.5 µl Tagment DNA enzyme I (Illumina 15027865), and incubated at 37C for 30 min on a heat block. For isolations resulting in under 30,000 microglia, microglia directly placed in 50 µl transposase reaction mix, as indicated above and incubated for 37C for 30 min. DNA was purified with Zymo ChIP DNA concentrator columns (Zymo Research D5205), eluted with 11 µl of elution buffer, and amplified using NEBNext High-Fidelity 2x PCR MasterMix (New England BioLabs M0541) with the Nextera primer Ad1 (1.25 µM) and a unique Ad2.n barcoding primer (1.25 µM) for 8-12 cycles. Libraries were size-selected through gel excision for fragments that were 175-255 bp and single-end sequenced for 51 cycles on a HiSeq 4000 or NextSeq 500.