title: GSM6720886: Adult, sample3, neocortex; Homo sapiens; RNA-Seq
library_construction_protocol: Tissues were dissociated by douncing 50-100 mg of prenatal tissue or 250 mg of adult tissue in 4 ml of lysis buffer (0.32 M sucrose, 5 mM calcium chloride, 0.1% Triton X-100, 0.1 mM ethylenediaminetetraacetic acid [EDTA], 3 mM magnesium acetate, and 1 mM dithiothreitol [DTT] in 1 mM Tris-HCl, pH 8.0) under RNase-free working conditions. Nuclei were isolated by sucrose gradient ultracentrifugation. RNA from single nuclei was prepared for sequencing using the 10X Genomics Chromium platform and the 3' gene expression (3' GEX) V3 kit, version 3.10. Each region and sample was barcoded and sequenced as separate libraries, without hashing. Nuclei were diluted and loaded with a target of 6,000 cells into nanoliter-scale Gel Bead-In-Emulsions (GEMs). Primers containing Illumina read 1 (R1) sequencing primers, a 16-bp 10x Barcode, a 10-bp randomer and a poly-dT primer sequence were subsequently mixed with the nuclear suspension and master mix. After incubation of the GEMs, barcoded, full-length cDNA was generated from pre-mRNA. Then, the GEMs were broken and silane magnetic beads were used to remove leftover biochemical reagents and primers. P5 and P7 primers, a sample index, and Illumina read 2 (R2) sequencing primers were added to each selected cDNA during end repair and adaptor ligation. P5 and P7 primers were used for Illumina bridge cDNA amplification (http://10xgenomics.com). Libraries were quantified using the Agilent Bioanalyzer and sequenced into 2x100 paired-end reads using the Illumina NovaSeq platform to target 50,000-100,000 reads per nucleus.