title: GSM5145420: WT NeuN+ H3K9me3 REP2; Mus musculus; ChIP-Seq
library_construction_protocol: Hi-C (Jiang, et. al.): Cortical tissue was prepped for fluorescence activated nuclear sorting (FANS) as follows. Briefly, the dissected tissue was homogenized in a hypotonic lysis solution and fixed in 1% formaldehyde for 10 minutes at room temperature. The cross-linking reaction was quenched with 125 mM glycine. The nuclei were then purified by centrifugation at 4000xg and resuspended in a 1:1 solution of the hypotonic lysis solution and a 1.8M sucrose solution prior to re-centrifugation at 4000xg to isolate out cortical nuclei. The pellet was then resuspended in Dulbecco's phosphate buffered saline (DPBS) containing 0.1% BSA and 1:1000 anti-NeuN antibody (clone A60, Alexa Fluor 488 conjugated; EMD Millipore Corp., MAB377X). Samples were incubated for 45 minutes while rotation and protected from light at 4C. DAPI (Invitrogen) was added immediately before sorting to label all nuclei. ChIP-seq: Cortical tissue was prepped for fluorescence activated nuclear sorting (FANS) as follows. Briefly, the dissected tissue was homogenized in a hypotonic lysis solution and fixed in 1% formaldehyde for 10 minutes at room temperature. The cross-linking reaction was quenched with 125 mM glycine. The nuclei were then purified by centrifugation at 4000xg and resuspended in a 1:1 solution of the hypotonic lysis solution and a 1.8M sucrose solution prior to re-centrifugation at 4000xg to isolate out cortical nuclei. The pellet was then resuspended in Dulbecco's phosphate buffered saline (DPBS) containing 0.1% BSA and 1:1000 anti-NeuN antibody (clone A60, Alexa Fluor 488 conjugated; EMD Millipore Corp., MAB377X). Samples were incubated for 45 minutes while rotation and protected from light at 4C. DAPI (Invitrogen) was added immediately before sorting to label all nuclei. Hi-C (Arima): Cortical tissue was prepped for fluorescence activated nuclear sorting (FANS) as follows. Briefly, the dissected tissue was homogenized in a hypotonic lysis solution and fixed in 1% formaldehyde for 10 minutes at room temperature. The cross-linking reaction was quenched with 125 mM glycine. The nuclei were then purified by centrifugation at 4000xg and resuspended in a 1:1 solution of the hypotonic lysis solution and a 1.8M sucrose solution prior to re-centrifugation at 4000xg to isolate out cortical nuclei. The pellet was then resuspended in Dulbecco's phosphate buffered saline (DPBS) containing 0.1% BSA and 1:1000 anti-NeuN antibody (clone A60, Alexa Fluor 488 conjugated; EMD Millipore Corp., MAB377X). Samples were incubated for 45 minutes while rotation and protected from light at 4C. DAPI (Invitrogen) was added immediately before sorting to label all nuclei. PacBio: Genomic DNA was isolated from FACS-sorted NeuN+ or NeuN- nuclei of adult mouse cerebral cortex using phenol chloroform extraction and was subsequently sheared to ~10kb using g-Tube microcentrifuge tubes (Covaris, 010145). RNA-Seq (RiboZero): Total RNA was first extracted from the prefrontal mouse cortices (n=12, 6WT/6KO), and prepared with RNeasy Lipid Tissue Mini kit (with on-column DNase1 treatment). The quantity and quality of RNA was checked using a bioanalyzer (Agilent RNA 6000 Nano Kit). RNA-Seq (QIAseq): Single-cell suspension from adult brain tissues was prepared using Miltenyi's Adult Brain Dissociation Kit (Miltenyi Biotec, 130-107-677) and Debris Removal Solution Kit (Miltenyi Biotec, 130-109-398) according to manufacturer instruction with minor modifications. In brief, total brain tissues, except olfactory bulb and cerebellum, were collected quickly and washed with ice cold 1x HBSS. After chopped into small pieces using sharp blade, the brain tissues were transferred into the C-tube containing 1950ul of Enzyme mix 1 and 30ul of Enzyme mix 2 from the kit and incubated on Miltenyi's gentle MACS Octo Dissociator with Heaters using program 37°C _ABDK_1 for 30 minutes. Afterwards, the digested tissue homogenate gently went through fire polished glass pippette 10 times, and then passed through a 70um cell strainer. Cells were collected via centrifuged at 300g for 10 minutes at 4 °C and resuspended in ice cold 1x HBSS. Debris Removal Solution was then added and overlayed with ice cold 1x HBSS. After centrifugation, three phases in the tube were clearly visualized, from top to bottom: 1x HBSS solution, debris and myelin layer, and single-cell suspension. Discard the top two phases completely, wash the cells in 1x HBSS, centrifuge to collect the cells, and then resuspended the cell pellet in 1 ml of 1x HBSS containing 1% fetal bovine serum. In order to minimize the unwanted microglia activation, single cell suspension was incubated in Fc receptor blocking Reagent (Miltenyi Biotec, 130-092- 575) for 10 min, and then the cell suspension was incubated with CD11b-microbeads (Miltenyi Biotec, 130-093-634) for 10 min at 4 °C in the dark, followed by positive selection with LS separation column (Miltenyi Biotec, 130-042-401). Flow cytometry analysis was performed to check the cell purity. The three different groups of cells (no enrichment, target and non-target cell population) were incubated in CD11b-FITC Monoclonal Antibody (M1/70) (eBioscience, 11-0112-81) at 1:2000 dilution for 30 min at 4 °C in the dark. Stained cells were examined using the Beckman flow cytometer. ATAC-seq: Single-cell suspension from adult brain tissues was prepared using Miltenyi's Adult Brain Dissociation Kit (Miltenyi Biotec, 130-107-677) and Debris Removal Solution Kit (Miltenyi Biotec, 130-109-398) according to manufacturer instruction with minor modifications. In brief, total brain tissues, except olfactory bulb and cerebellum, were collected quickly and washed with ice cold 1x HBSS. After chopped into small pieces using sharp blade, the brain tissues were transferred into the C-tube containing 1950ul of Enzyme mix 1 and 30ul of Enzyme mix 2 from the kit and incubated on Miltenyi's gentle MACS Octo Dissociator with Heaters using program 37°C _ABDK_1 for 30 minutes. Afterwards, the digested tissue homogenate gently went through fire polished glass pippette 10 times, and then passed through a 70um cell strainer. Cells were collected via centrifuged at 300g for 10 minutes at 4 °C and resuspended in ice cold 1x HBSS. Debris Removal Solution was then added and overlayed with ice cold 1x HBSS. After centrifugation, three phases in the tube were clearly visualized, from top to bottom: 1x HBSS solution, debris and myelin layer, and single-cell suspension. Discard the top two phases completely, wash the cells in 1x HBSS, centrifuge to collect the cells, and then resuspended the cell pellet in 1 ml of 1x HBSS containing 1% fetal bovine serum. In order to minimize the unwanted microglia activation, single cell suspension was incubated in Fc receptor blocking Reagent (Miltenyi Biotec, 130-092- 575) for 10 min, and then the cell suspension was incubated with CD11b-microbeads (Miltenyi Biotec, 130-093-634) for 10 min at 4 °C in the dark, followed by positive selection with LS separation column (Miltenyi Biotec, 130-042-401). Flow cytometry analysis was performed to check the cell purity. The three different groups of cells (no enrichment, target and non-target cell population) were incubated in CD11b-FITC Monoclonal Antibody (M1/70) (eBioscience, 11-0112-81) at 1:2000 dilution for 30 min at 4 °C in the dark. Stained cells were examined using the Beckman flow cytometer. Hi-C (Jiang, et. al.): Nuclei were digested with 100U MboI, and the restriction fragment ends were labeled using biotinylated nucleotides and re-ligated. After reversal of the cross-linking, ligated DNA was purified and sheared to a length of ~400bp by sonication, and the biotin-tagged ligation junctions were subsequently pulled down with streptavidin beads (Invitrogen, Dynabeads MyOne Streptavidin T1, Catalog No. 65602) and prepared into libraries. ChIP-seq: Nuclei were pelleted after FANS, resuspended in 300μl of micrococcal nuclease digestion buffer (10mM Tris pH 7.5, 4mM