title: GSM8152402: S26_CTE; Homo sapiens; RNA-Seq
library_construction_protocol: Nuclei isolation and sorting were performed on two donor samples per day, randomizing for diagnosis and age. Tissue was kept on ice throughout nuclei isolation. Tissue was homogenized and lysed in NST Buffer with DAPI (146mM NaCl, 10mM Tris, 1mM CaCl2, 21mM MGCl2, 0.1%BSA, 0.1% NP-40, 40U/ml Protector RNase Inhibitor, DAPI) and snipped with scissors on ice for 10 minutes. Debris was removed using a 70μm filter. Cells were spun down and resuspended in nuclei storage buffer (2% BSA, 400U/mL Protector RNase Inhibitor) to reach a concentration of 500-1000 nuclei/μL. Nuclei were purified for DAPI positive cells with a FACS-Aria flow cytometer to remove debris Library was performed according to the manufacturer's instructions (single cell 3' V2 protocol, 10xGenomics). Briefly, nuclei were combined with the master mix at the correct dilution and loaded with partitioning oil and gell beads into the chip to generate the gel bead emulsion or GEMs. Retrotranscription was performed to create cDNA from the RNA in the beads, with primer sequences and unique molecular identifier (UMI) barcodes, and the 10x genomics barcode included. Pooled barcoded cDNA was put through the 10x library prep protocol which includes amplification with PCR, SPRI bead cleanups for size selections and addition of paired end constructs and sample indices. single nucleus RNA sequencing