Single-nucleus RNA-seq

Single nucleus suspensions were isolated from ~ 50mg frozen human prefrontal cortex. Samples were homogenized in Nuclei EZ Lysis buffer (Cat#NUC101-1KT, Sigma-Aldrich) and incubated for 5 min. Samples were passed through a 70μm filter and incubated in additional lysis buffer for 5 min and centrifuged at 500 g for 5 min at 4°C before two washes in Nuclei Wash and Resuspension buffer (1xPBS, 1% BSA, 0.2U/μl RNase inhibitor). Nuclei were FACS sorted with DAPI (NucBlue Fixed Cell ReadyProbe Reagent, Cat#{"type":"entrez-nucleotide","attrs":{"text":"R37606","term_id":"795062","term_text":"R37606"}}R37606, Thermo) before running on the 10x Chromium™ Single Cell 3' v3 platform. cDNA library quantification and quality were assessed as in bulk RNA-seq. Libraries were sequenced using Illumina Novaseq 6000 S4 platform at the New York Genome Centre, using 100bp paired-end sequencing.