Sincle cell RNAseeq of homo sapien surgically resected brain tissue from mesial temporal lobe
Fresh-frozen postoperative and post-mortem brain tissues stored at -80 C were dissected in a cryostat chamber kept at constant -20 C to obtain samples of about 20-30 mg. When possible, a homogeneous mix of grey and white matter was dissected. Nuclei preparations were performed as previously described (Bizzotto et al, Science 2022, PMID  33737485). For some preparations, an intermediate step consisting of DAPI staining and fluorescence-activated nuclear sorting (FANS) was performed. Nuclei sorting was performed on a FACS Aria II cell sorter equipped with BD FACSDiva software, selecting all DAPI-positive nuclei (SSC-A and FSC-A gate boundaries were set at 10000 and 30, respectively). snRNA-seq was performed using the 10X Genomics Chromium Next GEM Single Cell 3-prime Reagent Kits. Around 30,000 nuclei were used to load the 10X Chromium, and were processed the same day for gel-bead in emulsion (GEM) generation, barcoding, and cDNA amplification, following manufacturer instructions. Libraries were sequenced (paired-end) on an Illumina NovaSeq6000 targeting 50,000 read-pairs per single-nucleus.