GSM7794024: sorted NeuN- nuclei from pooled prefrontal cortex white matter from middle aged (10 m) Olig2-SRF cKO male and female; Mus musculus; RNA-Seq
Prefrontal cortex was punched out using a 1.5mm biopsy punch (Hahn et al., 2023). Nuclei from two punches (one male and one female)  h were isolated with EZ Prep lysis buffer (Sigma, NUC-101) on ice following the manufacturer protocol. After removing supernatant, the pellet was resuspended with 4 ml chilled PBS and filtered through a 35-um cell strainer into a 5 ml round bottom FACS tube (Corning, 352235) and spun at 500g for 5min. After removing supernatant, nuclei were stained with anti-NeuN and anti-Olig2 and Hoechst. NeuN negative nuclei were sorted on a Sony800 to tubes. Nuclei were sorted and loaded on a standard Chromium Controller (10X Genomics) according to manufacturer protocols. We applied 12 PCR cycles to generate cDNA. Library construction was conducted using Chromium Single Cell 3' Library Construction Kit v3  using 14 PCR cycles for library generation (10X Genomics). All reaction and quality control steps were carried out according to the manufacturer's protocol and with recommended reagents, consumables, and instruments. Quality control of cDNA and libraries was conducted using a TapeStation (Agilent) at the Stanford Knight Initiative core.