title: GSM8891792: corpus callosum, normal chow, rep 2; Mus musculus; RNA-Seq
library_construction_protocol: Pooled tissue was transferred to chilled glass dounce with 1mL of lysis buffer + 1% TritonX100 + 1 μM DTT (Sigma Nuclei Pure Prep Kit, Cat #NUC-201). Samples were dounced on ice 10 times with pestle A, then 10 times with pestle B. The suspension was passed through a 40μM FlowMi cell strainer. Dounces were rinsed with 1mL of low sucrose buffer and passed through the cell strainer as well. Samples were centrifuged at 300 rcf, at 4°C, for 5 mins in a fixed-angle centrifuge. Supernatant was carefully decanted, and the pelleted nuclei were washed with 1.5mL of low sucrose buffer, and centrifuged again at 300 rcf, at 4°C, for 5 mins. Pelleted nuclei were then resuspended in 500μL Nuclei wash buffer (PBS + 1% UltraPure BSA + 0.2U/μL Protector RNase inhibitors) + 900μL of 1.8M sucrose cushion buffer, then carefully layered on top of 500μL 1.8M sucrose cushion buffer in a 2mL low-bind tube. Samples were centrifuged at 13,000 rcf, at 4°C, for 45 mins. Supernatant was removed, and nuclei were resuspended in 1mL of Nuclei wash buffer, passed through a 40μM FlowMi cell strainer and counted using a Cell Countess II. Approximately 6,000 nuclei were loaded per sample onto the 10X chromium controller per manufacturer's instructions. Single nuclei libraries were generated using the 10X single cell 3' reagents and protocol (v3.1). Libraries were sequenced on an Illumina Novaseq6000, with an average number of 80,000 reads per cell for each sample.