title: GSM3704365: 5-MAP2; Homo sapiens; RNA-Seq
library_construction_protocol: Methods for high-throughput isolation and profiling of single cells with neurofibrillary tangles (NFTs) and neighboring tangle-free neurons from fresh-frozen postmortem human brain: Tissue from the prefrontal cortex (Brodmann area 9; BA9) of Braak stage VI AD patients was thawed on ice, and the cortical ribbon spanning all cortical layers was micro-dissected. Gentle mechanical dissociation without detergents or enzymatic digestion followed by sucrose-iodixanol gradient centrifugation was used to purify cells with relatively well-preserved somas. Immunostaining and fluorescence-activated cell sorting using an antibody to detect pathological hyperphosphorylated tau (AT8) and the pan-neuronal marker MAP2 was used to collect two single-cell populations: AT8+ (either positive or negative for MAP2) and MAP2+/AT8-.  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.