Single-cell RNA-seq of Rat Cell Nuclei

RIN14B cells were put on ice, washed with chilled PBS, and then lysed with chilled Nuclei EZ lysis buffer (Sigma-Aldrich, NUC-101). Single cells were isolated with a 40 µm filter and pelleted in a centrifuge for 8 min, 800 rcf, 4°C. The nuclei were resuspended using PBS with 1% BSA and counted using a hemocytometer with trypan blue viability dye. The nuclei were centrifuged and resuspended at an appropriate volume for the 10X Chromium system (10X Genomics). The nuclei were counted once more to check the number and quality before proceeding with 10X Chromium processing and library construction as per the manufacturer’s instructions. Next Gen sequencing with a Chromium V2 chemistry was carried out on an Illumina NextSeq 500. Illumina NextSeq 500 pre-mRNA sequencing data were aligned to the Rattus norvegicus genome using CellRanger. The data were then analyzed with Seurat V3.0 as described previously (Butler et al., 2018 (link)).