GSM9112397: RNA_dUTP_Microglia_Clec7aPositiveCD229NegativeCD11cNegative_Cx3cr1Mef2aKO_Male_Adult_Cuprizone_5w_rep2_BC155_R1; Mus musculus; RNA-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). RNA-seq library preparation: Isolation and fragmentation of poly(A) and cDNA synthesis RNA-seq library preparation was performed similarly as previously described. Following sorting, 100,000 to 200,000 microglia were centrifuged (350 RCF at 4°C for 10 min), and resuspended into 100 µl lysis/Oligo d(T) Magnetic Beads binding buffer (Thermo Fisher Scientific A33562, or 100 mM Tris-HCl pH 7.5, 500 mM LiCl, 10 mM EDTA pH 8.0, 1% LiDS, 5 mM DTT, in molecular grade water) and stored at -80°C until processing. For isolation and enrichment of polyA mRNA, samples were thawed at RT and incubated with 20 µl Oligo d(T) Magnetic Beads (NEB S1419S) as follows: 2 min at 65°C on a PCR cycler and then 10 min at room temperature (RT). Samples were then placed on a collection magnet at RT and washed serially with 180 µl of washing buffer 1 (RNA-WB1; 10 mM Tris-HCl pH 7.5, 0.15 M LiCl, 1 mM EDTA pH 8.0, 0.1% LiDS, 0.1% Triton X-100, in molecular grade water) and then washing buffer 3 (RNA-WB3; 10 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA pH 8.0, in molecular grade water). RNA was eluted from Oligo (t) beads by resuspending the beads in 50 µl of elution buffer (EB; 10 mM tris-HCl pH 7.5, 1 mM EDTA pH 8.0, in water) followed by incubation at 80°C for 2 min on a PCR cycler. The samples were then placed back on the magnet, and the elution buffer supernatant containing the poly(A) RNA was carefully collected and placed on ice.  A second round of poly(A) RNA selection was then performed. Eluted Oligo d(T) beads were first washed on a magnet with 150 µl EB and then 150 µl 2X Oligo d(T) binding buffer (2x DTBB; 20 mM Tris-HCl pH 7.5, 1 M LiCl, 2 mM EDTA pH 8.0, 1% LiDS, 0.1% Triton X-100, in molecular grade water) and resuspended in 50 µl of 2x DTBB. Resuspended Oligo d(T) beads were then added to isolated RNA and incubated at 65°C and at RT as described above. Beads were then placed on a magnet and washed once with 180 µl of RNA-WB1, then once with 180 µl of RNA-WB3, and then resuspended in 30 µl of 1x First-Strand buffer (made from 5x stock Thermo Fisher Scientific 18080-044 with molecular grade water) transferred in a new PCR strip. Beads were collected on a magnet and resuspended in 10 µl of 2x SuperScript III buffer (made from 5x stock Thermo Fisher Scientific 18080-044 supplemented with 10 mM DTT, in molecular grade water). Resuspended beads were then incubated at 94°C for 9 min to fragment poly(A) RNA. Beads were then placed back on a magnet, and the supernatant containing poly(A) RNA was carefully collected and placed on ice.  Collected poly(A) mRNA in 2X SuperScript III buffer was then incubated for 1 min at 50°C on a PCR cycler with 2.5 µl of the following mix: 1.5 µg Random Primer (Thermo Fisher Scientific 48190-011), 10 µM Oligo d(T)20 primer (Thermo Fisher Scientific 18418020), 10 units SUPERase•In RNase Inhibitor (Thermo Fisher Scientific A32694), 4.0 mM dNTP mix (Thermo Fisher Scientific 18427088). Samples were then immediately placed on ice for 5 min. First-strand synthesis was then performed by incubation at 25°C for 10 min and 50°C for 50 min on a PCR cycler with 7.6 µl of the following mix: 0.2 µg actinomycin D (Sigma-Aldrich A1410; stock at 2 µg/µl in DMSO), 13.15 mM DTT (Thermo Fisher Scientific 361045836), 0.026% Tween-20 (from 10% stock Enzo 80-1929, in molecular grade water), 100 units SuperScript III Reverse Transcriptase (Thermo Fisher Scientific 18080-044), in molecular grade water. After incubation, RNA/DNA complexes were isolated by adding 36 µl of RNAClean XP beads (Beckman Coulter A63987) and incubated for 10 min at RT and then 10 min on ice. Samples were then placed on a magnet and beads were washed twice with 150 µl of 75% EtOH. Following washings, beads were air-dried at RT for 6-10 min and eluted with 10 µl of water.  Second-strand synthesis was then performed. RNA/DNA samples, in 10 µl of water, were incubated for 2.5 hours at 16°C with 5 µl of the following mix: 3x NEB Buffer 2 (10x stock New England Biolabs B7002S), 1.0 µl PCR mix (stock of 10 mM solution for each dATP, dCTP, dGTP, and dUTP from Thermo Fisher Scientific, in molecular grade water), 2.0 mM dUTP (Thermo Fisher Scientific, R0133), 1 unit RNAse H (Enzymatics Y9220L), 10 units DNA Polymerase I (Enzymatics P7050L), 0.03% Tween-20, in molecular grade water). DNA was then purified by addition of 1.5 µl Sera-Mag SpeedBeads Carboxyl Magnetic Beads (Thermo Fisher Scientific (Cytivia) 45152105050250), resuspended in 30 µl 20% PEG 8000/2.5 M NaCl (PEG 8000 from Sigma-Aldrich P2139; 5M NaCl 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).  RNA-seq final library preparation Sequencing libraries were prepared from generated cDNA 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).  RNA-seq libraries were 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 RNA-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.