Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Whole prefrontal cortex was dissected from 6-week old wild-type (WT) mice. Dissected tissue was thoroughly minced on ice using razor blades, then fixed with EGS for 30 min and/or for 5 min at room temperature with methanol-free formaldehyde (Pierce, Thermo Fisher Scientific) diluted to 1% in PBS. Fixation was quenched by adding 1/10th volume of 1.25 M glycine. Fixed tissue was collected by centrifugation at 200 g for 5 min at 4ºC, then washed twice with cold PBS. Washed tissue was flash-frozen with liquid nitrogen then cryofractured using a Covaris CryoPrep Impactor on power setting 6. Nuclei and chromatin immunoprecipitation were performed as in Markenscoff-Papadimitriou et al.(Markenscoff-Papadimitriou et al., 2014): briefly, the cryofractured tissue was lysed in ChIP Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 nM NaCl, 0.5% NP-40, 0.25% Sodium Deoxychoalate, 0.1% SDS) in rotation for 40 min at 4ºC. Nuclei were centrifuged (1700x g, 5 min, 4ºC), and the pellet was resuspended in shearing buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8, 0.25% SDS), then sheared on a Covaris S2 sonicator (12 or 16 min, 2% Duty Cycle, Intensity 3, 200 cycles per burst, frequency sweeping). Sheared chromatin was centrifuged (10,000 g for 10 min at 4ºC) to remove insoluble material. Sheared chromatin was diluted 5-fold with ChIP Dilution Buffer (CDB: 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS) and pre-cleared for two hours at 4ºC with protein G dynabeads (Thermo Fisher Scientific). Each ChIP was set up with the appropriate antibody (10 mg of anti-Setd1a, 1 mg of anti-H3K27ac, -H3K4me2 and -H3K4me3, 3 mg of anti-H3K4me1, 5 mg of Mef2) and 10-20 mg of cleared chromatin, incubating then overnight at 4ºC. Protein G dynabeads were blocked overnight with 2 mg/ml yeast tRNA (Thermo Fisher Scientific), then added to antibody bound chromatin and rotated for 3 hr at 4ºC. Bead-bound chromatin was washed 5 times with LiCl Wash Buffer (100 mM Tris-HCl pH 7.5, 500 mM LiCl, 1% NP-40, 1% Sodium Deoxycholate) and once with TE (pH7.5). DNA was eluted from beads by incubating at 65ºC for 30 min with 25 mL ChIP Elution Buffer (1% SDS, 0.1 M Sodium Bicarbonate). This elution was repeated and the combined elution fractions were incubated overnight at 65ºC with proteinase K (Thermo Fisher Scientific) and RNaseA (Thermo Fisher Scientific). ChIP DNA was purified 1.8X of AMPure XP beads and eluted in 30 ml of nuclease free water. Libraries were prepared with Ovation Ultralow System V2 1-16 (NuGEN technologies) according to manufacturers' recommendation. Total RNA was extracted from prefrontal cortices of 6-week-old mice using RNeasy Mini Kit (Qiagen). We analyzed each four male Setd1a+/- mice and their wild-type (WT) male littermates. Quality of RNA samples was assessed by Bioanalyzer (Agilent). Mean (± SD) RNA integrity number (RIN) was 9.3 (± 0.5) in the mutant mice and 9.7 (± 0.2) in WT. An RNA-seq library was prepared from ~400 ng of total RNA by using TruSeq RNA prep kit (Illumina). Messenger RNAs were enriched by poly-A selection. Libraries were sequenced on HiSeq2500/HiSeq4000 (Illumina) with single-end 100bp reads at Columbia Genome Center. The tissue was incubated for 1h at 37°C on a rocking platform with papain-EBSS (1 PFC/mL). The tissue was triturated 20 times and cells were pelleted (400 g, 5 min, RT). Remaining papain was inhibited by resuspending the cell pellet with Ovomucoid protease inhibitor solution diluted 1:10 in EBSS. An extra 5 ml of Ovomucoid protease inhibitor solution was slowly layered at the bottom of the cell suspension to create a discontinuous gradient and cells were pelleted (100 g, 6 min, RT). Cells were resuspended in sort media supplemented with 100 U/mL DNase I (Worthington Biochemical), 4 mM MgCl2, and 500 ng/mL DAPI (Invitrogen). These cells were passed through a 40 uM cell strainer, and then FAC sorted. Live cells were selected by gating out DAPI positive cells and positive cells were gated by tdTomato fluorescence. Fifty thousand cell were used to prepare ATAC-seq libraries as described in references (Buenrostro et al., 2015; Monahan et al., 2017). In brief, cells were pelleted (500 g, 5 min, 4°C) and resuspended in ice cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Nuclei were pelleted (1000 g, 10 min, 4°C) and resuspended in transposition reaction mix prepared from Illumina Nextera reagents (for 50 μL: 22.5 μL water, 25 μL 2xTD buffer, 2.5 μL Tn5 Transposase). Transposed DNA was column purified using a Qiagen MinElute PCR cleanup kit (Qiagen) and amplified using barcoded primers and NEBNext High Fidelity 2x PCR Master Mix (NEB). Amplified libraries were purified using Ampure XP beads (Beckman Coulter). Cells were dissociated using the papain dissociation kit (Worthington) following the manufacturers' recommendation. In brief, the PFC of 2 WT and 2 Setd1a+/- mice was dissected and transferred to ice-cold PBS. The PFC was cut in small pieces using a razor blade and dissociated with a papain dissociation system (Worthington Biochemical). The tissue was incubated for 1h at 37°C on a rocking platform with papain-EBSS (1 PFC/mL). The tissue was triturated 20 times and cells were pelleted (400 g, 5 min, RT). Remaining papain was inhibited by resuspending the cell pellet with Ovomucoid protease inhibitor solution diluted 1:10 in EBSS. An extra 5 ml of Ovomucoid protease inhibitor solution was slowly layered at the bottom of the cell suspension to create a discontinuous gradient and cells were pelleted (100 g, 6 min, RT). To enrich for living cells, we removed dead cells from single cell suspension using the MACS® Dead Cell Removal Kit (Miltenyi Biotec) with the MS Columns following manufacturer's recommendation. In brief, 200 μl per PFC of Dead Cell Removal MicroBeads were added the pelleted cells. Cell were resuspended and incubated for 15 min at RT. The MS columns were washed once with 0.5 ml of Binding buffer. After incubation is complete, the cell suspension was diluted with 500 μl 1X Binding Buffer and the suspension was applied to the MS columns. The flowthough was collected. To increase the number of live cells recovered, the column was washed twice with 1 ml of Binding buffer at RT. Flowthough were combined and live cell were pelleted (400 g, 5 min, RT). Cells were then washed twice with PBS containing 0.04% BSA and the final pellet was resuspended in 300 ml of PBS containing 0.04% BSA and passed through a 40 uM cell strainer. Cell were counted and viability was evaluated by trypan blue staining.The scRNA-Seq was performed using the 10x Genomic platform and the “Single Cell 3' v2” by the Columbia Genome Center.