GSM9112476: ChIPseq_InputH3K27me3_Microglia_WT_Male_Adult_Basal_rep3_BC105_R1; Mus musculus; ChIP-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). Chromatin immunoprecipitation Following sorting, aliquots of 400,000 to 600,000 microglia were centrifuge (1000 RCF at 4°C for 10 min), and cell pellet were snap-frozen in liquid nitrogen and stored at -80°C. When ready, chromatin immunoprecipitation for histone modification H3K27me3 was performed as follows, using microglia 400,000 to 600,000 per replicates. First microglia were briefly thawed on ice and lysed by incubation in 1 ml of lysis buffer (0.5% IGEPAL CA-630, 10 mM HEPES pH 7.9, 85 mM KCl, 1 mM EDTA pH 8.0, in molecular grade water) for 10 min on ice. Lysates were centrifuged at 800 RCF for 5 min at 4°C, and pellets were resuspended in 200 µl of sonication/ immunoprecipitation buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EGTA, 0.1% Deoxcycholate, 0.5% Sarkosyl, in molecular grade water). Sonication was performed with a Bioruptor Standard Sonicator (Diagenode) and consisted of two rounds of 10 cycle of 30 sec “sonication-on”, 90 sec “sonication-off”.  Twenty-two microliters of 10% Triton-X were then added to samples (1% final concentration) on ice, and lysates were cleared by centrifugation for 5 min at 18,000 RCF at 4°C. Two microliters of supernatant were then set aside for input library sequencing controls. Each sample supernatants were then immunoprecipitated with 5.0 µg H3K27me3 antibody (Active Motif 39155) pre-bound to 17 µl Protein A Dynabeads (Thermo Fisher Scientific 10001D), on a rotator for three hours at 4°C.  Immunoprecipitates were washed three times each on ice with ice-cold wash buffer I (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA pH 8.0 in molecular grade water), wash buffer III (10 mM Tris-HCl, 250 mM LiCl, 1% IGEPAL CA-630, 0.7% Deoxycholate, 1mM EDTA in water) and TET (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 0.1% Tween-20, in molecular grade water) and eluted with 1% SDS/TE at RT in a final volume of 100 µl.  Reverse-crosslinking was then performed on immunoprecipitates and saved input aliquots. First, 6.38 µl of 5 M NaCl (final concentration 300 mM; Thermo Fisher Scientific AM9759) was added to each sample immersed in 100 µl SDS/TE. Crosslinking was then reversed by overnight incubation at 65-68°C in a hot air oven. Potentially contaminating RNA was then digested for one hour at 37°C with 0.33 mg/ml RNase A, proteins were digested for one hour at 55°C with 0.5 mg/ml proteinase K (NEB P8107S), and DNA was extracted using Sera-Mag SpeedBeads (Thermo Fisher Scientific 6515205050250).  DNA was then purified by addition of 1.5 µl Sera-Mag SpeedBeads Carboxyl Magnetic Beads (Cytivia 45152105050250), resuspended in 150 µl 20% PEG 8000/2.5 M NaCl (PEG 8000 from Sigma-Aldrich P2139; NaCl 5M from Thermo Fisher Scientific AM9759), incubated at RT for 15 min and placed on a magnet for two rounds of bead washing with 80% EtOH. Beads were then air dried for 6-10 min and DNA was eluted from the beads by adding 40 µl of water. Supernatant was then collected on a magnet and placed on ice or stored at -20°C until DNA blunting, poly(A)-tailing, and adapter ligation (see below).  ChIP-seq final library preparation Sequencing libraries were prepared from recovered DNA (ChIP) by blunting, A-tailing, and adapter ligation using barcoded adapters (NEXTflex Unique Dual Index Barcodes (Set B)(Bioo Scientific NOVA-514151), as described in Gosselin et al. 2014. Libraries were PCR-amplified for 14 cycles and size selected for fragments (200-400 bp for ChIP-seq) by gel extraction (10% TBE gels, Thermo Fisher Scientific Technologies EC62752BOX).  ChIP-seq libraries were single- or 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 ChIP-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.