single-cell ATAC-seq for speciment F06, sample GW006, isolated Microglia ex vivo
Sorted microglia were centrifuged for 5 minutes at 300 rcf and the supernatant was carefully aspirated. Cells were permeabilized using lysis buffer (10 mM Tris-HCl pH 7.4 (15567027, Thermo Fischer Scientific), 10 mM NaCl (ICN15194401, Fischer Scientific), 3 mM MgCl2 (194698, Mp Biomedicals Inc.), 0.1% Tween 20 (P7949, Sigma), 0.1% IGEPAL-CA630 (I8896, Sigma), 0.01% Digitonin (G9441, Promega), and 1% BSA in nuclease free water) and incubated for 5 minutes on ice. Permeabilized nuclei were washed using washing buffer (lysis buffer without IGEPAL-CA630 and Digitonin), centrifuged for 5 minutes at 500 rcf and resuspended in Nuclei buffer (10x Genomics). An aliquot was mixed with Trypan Blue and counted using a hemocytometer. Up to 15,300 nuclei were tagmented before loading onto a Chromium Controller; libraries were generated according to manufacturer specifications ((Chromium Next GEM Single Cell ATAC Library and Gel Bead Kit v1.1, 1000175; Chromium Next GEM Chip H Single Cell Kit, 1000162; Single Index Kit N Set A, 1000212, 10x Genomics)). Libraries were amplified for 10 PCR cycles. Libraries were sequenced using a NovaSeq 6000 (Illumina) using these read lengths: Read 1: 50 cycles, Read 2: 50 cycles, Index 1: 8 cycles, Index 2: 16 cycles.