Name: GSM7040224
Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC SINGLE CELL
Selection: cDNA
Layout: PAIRED
Construction protocol: Nuclei were isolated from 24 samples of frozen human postmortem brain tissue. Samples weighing 40mg were homogenized in 5mL glass douncers with 1mL lysis buffer containing DAPI using glass pestles using ~10 strokes per pestle. Homogenate was filtered using a 40um mesh strainer (FisherScientific #48680). Nuclei were sorted for DAPI fluorescence using a BD FACS Aria at the Boston University Flow Cytometry Core. Approximately 100,000 nuclei were sorted into 7ul of 0.04% bovine serum albumin (MilliporeSigma #126615) in phosphate buffered saline (ThermoFisher #10010031). Nuclei were counted using a hemocytometer and assessed for concentration and debris. 7,000 nuclei were targeted per sample except for two samples with lower concentrations where 5,000 nuclei were targeted. The 10x Chromium process was performed and next generation sequencing libraries were prepared using the 10x genomics single cell 3' gene expression dual index kit. Libraries were sequenced at the Boston University Single Cell Sequencing Core. The pool of snRNA-seq libraries were sequenced on 7 Next-seq P3 flow cells with intermediate re-pooling scheme to optimize for 50-80% sequencing saturation, > 8,000 average UMI per cell. Between sequencing runs, we preliminarily aligned the sequencing reads as outlined below to assess quality per sample and estimate the number of viable nuclei and sample complexity. We identified two samples, C-13291 and C-612, to have low quality QC metrics due to wetting failures, mean UMI per cell <1,000 and estimated # cells >50,000. These samples were excluded from subsequent re-pooling and further analyses (Supplemental Table 1.3 tab STARsolo QC). snRNA-seq