title: GSM5066668: ATAC_Microglia_Ki67Positive_Ki67rfp_Male_PND75_PLX5622_17d_Recovery_3d_rep1; Mus musculus; ATAC-seq
library_construction_protocol: Microglia isolation: Mice were first euthanized with an overdose of ketamine-xylazine administered by intra-peritoneal injection and subsequently perfused transcardially with PBS. Brain were then removed from the skull and immediately homogenized in a buffer solution (HBSS (Life Technologies, 14175-095), 1% BSA (Fisher), 1mM EDTA (Invitrogen), 1 mM sodium butyrate (Sigma B5887-1G), 1 uM Flavoperidol (Sigma F3055) by gentle mechanical dissociation on ice, using a 2 ml polytetrafluoroethylene pestle (Wheaton, 358026) as performed in Gosselin et al. 201417. Microglia were then enriched by Percoll (GE) gradient, washed and labeled with Live/Dead yellow dead stain (ThermoFisher), then incubated 1:100 with a CD16/CD32 receptor blocking antibody (1:50, eBioscience 14-0161, clone 93) for 20 min, and then incubated 1:100 with antibodies directed against cell surface proteins CD11b-BV421 (BioLegend 101251, clone M1/70), CD45-APC (BioLegend 103112, clone 30-F11), and CD44-APC-Cy7 (BioLegend 103028, clone IM7). For all ChIP-seq experiments, microglia were then further fixed with 1% PFA for 10 min at RT, permeabilized with reagents from ebioscience Foxp3 Transcription Factor staining buffer set (ThermoFisher 00-5523-00) and stained with anti-Ki67-Alexa 488 antibody (BD Pharmingen 561165, clone B56). Microglia were either analyzed with BD LSR or sorted with BD Aria II instruments (100 um nozzle). Flow cytometry results were analyzed using FlowJo software (Tree Star). RNA-seq library preparation: Isolation and fragmentation of poly(A)   For RNA-seq, we generated 2 independent biological replicates per conditions (n=2). Following sorting, 200,000 microglia were pelleted and put into 100 ul lysis/Oligo d(T) Magnetic Beads binding buffer (100 mM Tris-HCl ph 7.5, 500 mM LiCl, 10 mM EDTA pH 8.0, 1% LiDS, 5 mM DTT, in water) and stored at -80C until processing. For human brain cortical RNA, 500 ng of RNA was diluted with proper volume of 2x lysis/ Oligo d(T) Magnetic Beads binding buffer to a final concentration of 1x lysis/Oligo d(T) Magnetic Beads binding buffer. For sequencing library preparation, samples were incubated with 20 ul Oligo d(T) Magnetic Beads (NEB, S1419S) in PCR strips for selection of polyadenylated (poly(A)) mRNA 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 ul 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 water) and then washing buffer 3 (RNA-WB3; 10 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA pH 8.0, in water). RNA was eluted from Oligo (t) beads by resuspending the beads in 50 ul of elution buffer (EB; 10 mM tris-HCl pH 7.5, 1 mM EDTA pH 8.0, in water) followed by incubation at 80C 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 ul EB and then 150 ul 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) and resuspended in 50 ul 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 150 ul of RNA-WB1 and then with 30 ul of 1x First-Strand buffer (made from 5x stock; Life Technologies) and transferred in a new PCR strip. Beads were collected on a magnet and resuspended in 10 ul of 2x SuperScript III buffer (Life Technologies, 1x First-Strand buffer, 10 mM DTT in 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.  RNA-seq library preparation: Synthesis of cDNA  RNA in 2X SuperScript III buffer was incubated for 1 min at 50 C on a PCR cycler with 2.5 ul of the following mix: 1.5 ug Random Primer (Life Technologies 48190-011), 10 uM Oligo d(T) (Life Technologies 18418020), 10 units SUPERase-In (Ambion AM2696), 4 mM dNTP mix (Life Technologies 18427088), in water. 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 ul of the following mix: 0.2 ug actinomycin (Sigma A1410), 13.15 mM DTT (Life Technologies), 0.026% Tween-20, 100 units SuperScript III (Life Technologies kit 18080-044), in water. After incubation, RNA/DNA complexes were isolated by adding 36 ul of Agencourt 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 ul of 75% EtOH. Following washings, beads were air-dried for 10-12 min and eluted with 10 ul of water.  Second-strand synthesis was then performed. RNA/DNA samples in 10 ul of water were incubated for 2.5 hours at 16 C with 5 ul of the following mix: 3x Blue Buffer (Enzymatics), 1.0 ul PCR mix (Affymetrix 77330), 2.0 mM dUTP (Affymetrix 77206), 1 unit RNAseH (Ezymatics Y9220L), 10 units DNA Polymerase I (Enzymatics P7050L), 0.03% Tween-20, in water). DNA was then purified by addition of 1.5 ul Sera-Mag SpeedBeads (Thermo Scientific, 651520505025), resuspended in 30 ul 20% PEG 8000/2.5 M NaCl, 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 10-12 min and DNA was eluted from the beads by adding 40 ul of water. Supernatant was then collected on a magnet and placed on ice or stored at -20C until DNA blunting, poly(A)-tailing, and adapter ligation (see below).   RNA-seq final library preparation  Sequencing libraries were prepared from generated cDNA (RNA) by blunting, A-tailing, and adapter ligation as previously described using barcoded adapters (NextFlex, Bioo Scientific). Prior to final PCR amplification, RNA-seq libraries were digested by 30 min of incubation at 37C with Uracil DNA Glycosylase (final concentration of 0.134 units per ul of library volume; UDG, Enzymatics G5010L) to generate strand-specific libraries. Libraries were PCR-amplified for 12-15 cycles and size selected for fragments (200-500 for RNA-seq) by gel extraction (10% TBE gels, Life Technologies EC62752BOX). RNA-seq libraries were single-end sequenced for 76 cycles on an Illumina HiSeq 4000 (Illumina, San Diego, CA) according to manufacturer's instruction. Chromatin immunoprecipitation: Chromatin immunoprecipitation for histone modification H3K4me3, H3K27ac and Rpb2 and was performed as follows, using ~500,000 microglia per assay and n=2 independent biological replicates per conditions. 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 water) for 10 min on ice. Lysates were centrifuged at 800 RCF for 5 min at 4 °C, and pellets were resuspended in 200 ul 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 water). Sonication was performed with a Bioruptor Standard Sonicator (Diagenode) and consisted of two rounds of 15 min each, alternating stages of 30 sec “sonication-on” with 60 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. Supernatants were then immunoprecipitated on a rotator for two hours at 4 °C with antibody pre-bound to 17 ul Protein A Dynabeads (Life Technologies 10001D). The following antibodies were used: H3K4me3 (Millipore