GSM7063462: ATAC-Microglia, SALL1 Enhancer KO, Rep2; Mus musculus; ATAC-seq
Mouse brains were homogenized as previously described by gentle mechanical dissociation (Gosselin et al, 2014) and stained with anti-mouse anti-CD11b-APC (BioLegend 101212), anti-CD45-Alexa488 (BioLegend 103122), and anti-CX3CR1-PE (BioLegend 149006). Microglia were sorted as DAPI negative, singlets, and CD11b+CD45lowCX3CR1+. Approximately 50,000 sorted microglia were prepped for ATAC-seq as previously described (Buenrostro et al., 2013). Briefly, cells were lysed in 150 uL lysis buffer (10mM 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 uL transposase reaction mix (1x Tagment DNA Buffer (Illumina 15027866), 2.5 uL DNA enzyme I (Illumina 15027865), and incubated at 37°C for 30 minutes. DNA was purified with Zymo ChIP DNA concentrator columns (Zymo Research D5205), eludated with 11 uL of elution buffer, and amplified using NebNext High-fidelity 2x PCR MasterMix (New England BioLabs M0541) with the Nextera primer Ad1 (1.25 uM) and a unique Ad2.n barcoding primer (1.25 uM) for 8-12 cycles  Resulting libraries were size selected by gel excision to 155-250 bp, purified, and single end sequenced using a HiSeq 4000 (Illumina) for 51 cycles according to the manufacturer's instructions.