title: GSM3704373: 6b-MAP2; Homo sapiens; RNA-Seq
library_construction_protocol: Comparison of single transcriptomes from neuronal nuclei versus somas, isolated using the pan-neuronal markers NeuN (nuclear) and MAP2 (cytoplasmic): Tissue from the prefrontal cortex (Brodmann area 9; BA9) of an AD Braak stage VI patient (case # 6) was thawed on ice, and the cortical ribbon spanning all cortical layers was micro-dissected. Dissociation of individual nuclei used mechanical force and a detergent (0.1% triton); dissociation of individual somas used gentle mechanical force. Immunostaining and FACS using the pan-neuronal markers NeuN or MAP2 was used to collect three populations: MAP2+, NeuN+, and All nuclei (NeuN+ and NeuN-). RNA capture and library preparation was performed using the 10× Genomics Chromium Single Cell 3' v2 assay. The generated paired-end libraries were sequenced on Illumina Novaseq 6000. Single-cell RNA capture and library preparation was performed using the 10× Genomics Chromium Single Cell 3' v2 assay according to the manufacturer's user manual. The numbers of cells loaded ranged from 1,400–11,000 to capture the maximum number of cells up to an upper limit of ~5,000 cells per sample (for an expected cell capture efficiency of ~40%). For cDNA amplification, the number of PCR cycles was 13–15 (adjusted to the targeted cell recovery). For library construction, the number of cycles for the sample index PCR reaction was 12–13 (adjusted to the quantified cDNA input). The generated paired-end libraries were sequenced on an Illumina Novaseq 6000 (2 × 100); 26 bp for read 1 (cell barcode and UMI), 8 bps for the i7 index, and 98 bps for read 2.