GSM9112469: ATACseq_Microglia_Clec7aPositiveCD229PositiveCD11cPositive_Cx3cr1Mef2aKO_Male_Adult_Cuprizone_5w_rep2_BC17_R1; Mus musculus; ATAC-seq
Mice were euthanized with an overdose of ketamine-xylazine solution (90 ml/kg & 10 ml/kg) injected intraperitoneally and subsequently perfused transcardially with ice-cold 0.9% NaCl saline solution (Sigma-Aldrich, S5886-1K6). Brains were extracted and placed in ice-cold base buffer (BB) solution (HBSS 1X (Life Technologies, 14175-095), 1% BSA (Sigma-Aldrich, A9647), 1mM EDTA (Thermo Fisher Scientific, 15575020), 1 mM sodium butyrate (Sigma-Aldrich, B5887), 1 mM Flavoperidol (Sigma-Aldrich, F3055))35. For flow cytometry, brains were cut into smaller pieces in BB solution before adding Accutase solution (Sigma-Aldrich, A6964) to enzymatically dissociate the brain. After 1 hour of incubation, brains were further homogenized on ice using a cut P1000 tip, then an uncut tip and finally a P200 tip. For FACS, brains from 2-6 mice were from same conditions were pooled and homogenized in BB solution by gentle mechanical dissociation on ice using a 7 ml polytetrafluoroethylene pestle (Duran Wheaton Kimble, 357542) and filtered through a 100 µm cell strainer (ThermoFisher 22-363-549).  Following brain homogenization, homogenates were resuspended in 37% Percoll (Thermo Fisher Scientific (Cytivia) 17089101; 8 ml/brain, in 15 ml tubes (1 tube/brain), which were then centrifuged at 600 x g for 20 min at 20°C, with medium acceleration and no deceleration. Pellets were then recovered and washed once in with BB solution, centrifuged at 300 RCF at 4°C for 7 min, resuspended in 4 ml HBSS 1X and then centrifuged at 300 RCF at 4°C for 7 min to remove proteins. Cells were resuspended in 100 µl HBSS1x and incubated on ice in the dark for 10 min with 1:100 anti-CD16/CD32 receptor unconjugated antibody (BioLegend 101302, clone 93). Then, cells were incubated on ice in the dark for 10 min with 1:1000 Live/Dead yellow stain (Thermo Fisher Scientific L34959) and conjugated antibodies directed against cell surface proteins: 1:167 anti-Ace-Alexa 750 (R&D Systems FAB15131S, clone 230214), 1:100 anti-CD11b-Brilliant Violet 421 (BioLegend 101251, clone M1/70), 1:100 anti-CD44-APC/Cy7 (BioLegend 103027, clone IM7), 1:200 anti-CD45-BB700 (BD Biosciences 566439, clone 30-F11), 1:100 anti-CD229-Alexa 647 (BD Biosciences 566689, clone Ly9.7.144), 1:133 anti-CD11c-PE (BioLegend 117308, clone N418) and 1:100 anti-Clec7a-PE/Cy7 (Thermo Fisher Scientific 25-5859-80, clone bg1fpj). After incubation, cells were washed in HBSS1x and centrifuged at 300 RCF at 4°C for 7 min. Then, cell pellets were either 1) resuspended in BB and filtered through a 35 µm cell strainer and sorted for RNA-seq and ATAC samples, or 2) underwent cross-linking for ChIP-seq samples. For cross-linking, cell pellets were resuspended in HBSS 1x and fixed with 1% PFA at RT in the dark for 10 min, after which 1/20th volume of 2.625 M glycine (Thermo Fisher Scientific BP381-500) was added for quenching for 10 min. Cells were washed in BB, centrifuged at 1000 RCF at 4°C for 10 min and filtered through a 35 µm cell strainer using BB and then sorted. Flow cytometry data was acquired with a BD FACSymphonyA1 instrument. Data was analyzed with FlowJo (v10.10.0). Microglia were sorted with BD FACSAria Fusion instrument (100 µm nozzle). Assay for Transposase-Accessible Chromatin-sequencing (ATAC-seq) Immediately after sorting, aliquot of 80,000 microglia were centrifuged (350 RCF at 4°C for 10 min) and cell pellets 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 molecular grade water) on ice and nuclei were pelleted by centrifugation at 500 RCF for 10 min. Nuclei were then gently resuspended on ice in 50 µl transposase reaction mix (1x Tagment DNA buffer (Illumina 15027866), 2.5 µl Tagment DNA enzyme I (Illumina 15027865), in molecular grade water) and incubated at 37C for 30 min on a PCR cycler. DNA was then purified with Zymo ChIP DNA concentrator columns (Zymo Research D5205) and eluted with 10 µl of elution buffer. DNA was then amplified with PCR mix (1.25 µM for each of i5- and i7-indexed primers, from Mezger et al 2018, 0.6x SYBR Green I (Thermo Fisher Scientific S7563), 1x NEBNext High-Fidelity 2x PCR MasterMix, (NEB M0541)) for 9 cycles, run on gel for size selection of fragments (160-280 bp) as detailed above, and paired-end sequenced for 100 or 150 cycles on an Illumina NovaSeq X Plus or NovaSeq 6000 (Illumina, San Diego, CA) according to manufacturer's instruction.  Illumina sequencing Sequencing for ATAC-seq samples was performed on NovaSeq X Plus at the IGM Genomics Center, University of California, San Diego, La Jolla, CA, USA, or NovaSeq 6000 at The Centre for Applied Genomics (TCAG) at The Hospital for Sick Children, Toronto, ON, Canada.