Mab377x

Nuclei were isolated from pulverized postmortem brain tissue (0.7 g) using sucrose gradient centrifugation (4 °C, 25000 rpm, 1 h) and incubated with anti-NeuN-488 antibody (Millipore MAB377X, 1:1000) in a phosphate buffered saline (PBS) solution containing bovine serum albumin (BSA at 0.1%). Nuclei were sorted into NeuN+ and NeuN− fractions by FACS (BD FACSAria™III). Fibroblasts were cultured in fibroblast culture media (DMEM high glucose, FBS, l-glutamine, N.E. amino acids, Pen/Strep; all Invitrogen) and confluent fibroblasts were harvested for gDNA isolation. DNA was extracted from: (i) 0.3 g post-mortem bulk brain tissue; (ii) NeuN+ and NeuN− nuclear fractions; and (iii) fibroblast cell lines using the DNeasy Blood and Tissue Kit (QIAGEN) that included an RNAse A treatment step (QIAGEN) according to protocols provided by the manufacturer. For all samples, the libraries were prepared using the Illumina TruSeq DNA PCR-Free Library Prep Protocol with 1.5 μg of DNA from NeuN+ and NeuN− nuclear fractions and 3 μg of DNA from bulk cortex and fibroblast samples. Then, 150 bp paired-end reads were generated using the Illumina HiSeq X platform. The sequencing experiments were designed to yield 90× coverage. Library construction and sequencing were conducted at Psomagen, previously known as Macrogen (Rockville, MD, USA).

Nuclei were isolated as previously described^{1 (link),6 (link)}. Isolated nuclei were labelled by incubation with 1:1,000 dilution of Alexa Fluor 488-conjugated anti-NeuN antibody (MAB377X, Millipore) and a 1:1,000 dilution of Hoechst 33342 at 4 °C for 1 h with continuous shaking. FANS of single nuclei was performed using a BD Influx sorter with an 85-μm nozzle at 22.5 PSI sheath pressure. Single nuclei were sorted into each well of a 384-well plate preloaded with 2 μl of proteinase K digestion buffer (1 μl M-Digestion Buffer (Zymo, D5021-9), 0.1 μl of 20 μg μl⁻¹ proteinase K and 0.9 μl H₂O). The alignment of the receiving 384-well plate was performed by sorting sheath flow into wells of an empty plate and making adjustments based on the liquid drop position. Single-cell (one-drop single) mode was selected to ensure the stringency of sorting. For each 384-well plate, columns 1–22 were sorted with NeuN⁺ (488+) gate, and column 23-24 with NeuN⁻ (488−) gate, reaching an 11:1 ratio of NeuN⁺ to NeuN⁻ nuclei. BD Influx Software v1.2.0.142 was used to select cell populations.

Brain nuclei were isolated as previously described^{13 (link),18 (link)}, with initial homogenization performed with either 1% formaldehyde in Dulbecco’s phosphate-buffered saline or 2 mM DSG (disuccinimidyl glutarate) (ProteoChem) in Dulbecco’s phosphate-buffered saline. Nuclei were stained overnight with PU.1-PE (Cell Signaling 81886S, 1:100), OLIG2-AF488 (Abcam 225099, 1:2,500) or SALL1 AF647 (Thermo Fisher, clone NRNSTNX 51-9279-82, 1:100) or NEUN-AF488 (Millipore MAB 377X, 1:500). Nuclei were washed the following day with 4 ml FACS buffer, passed through a 40 µM strainer, and stained with 0.5 μg ml⁻¹ DAPI. Nuclei for each cell type were sorted with a Beckman Coulter MoFlo Astrio EQ cell sorter and pelleted at 1,600g for 5 min at 4 °C in FACS buffer. Nuclei pellets were snap frozen and stored at −80 °C before library preparation.

Freshly isolated mouse brains were homogenized in nuclear isolation buffer (10 mM tris-HCl, 50 mM sodium disulfite, 1% Triton X-100, 10 mM MgCl₂, and 8.6% sucrose, pH 6.5) and centrifuged for 5 min at 455g at 4°C. The resulting pellet was washed four times with 900 μl of nuclear isolation buffer with centrifugation at 455g at 4°C. The pelleted nuclei were then resuspended in 1.4 ml of PBS, filtered through a cell strainer, and stained with a 1:1000 dilution of the anti-NeuN antibody (clone A60, Alexa Fluor 488–conjugated, Millipore MAB377X) for 45 min by rotation in the dark at 4°C after adding bovine serum albumin (BSA) to a final concentration of 0.1%. After washing in PBS, the stained nuclei were filtered through FACS tubes, sorted on a FACS Aria III (BD Biosciences) sorter in PBS, pelleted 5 min at 590g at 4°C, and the pellet was immediately used for RNA isolation or frozen at −80°C.