GSM8606575: Control hippocampus, postnatal day 19, snRNAseq; Mus musculus; RNA-Seq
Nuclei were isolated using 10x Genomics Chromium Nuclei Isolation Kit (10x Genomics, Cat# 1000493) lysis buffer and the debris was removed using Miltenyi Anti-Nucleus beads (Miltenyi Biotec, Cat# 130-132-997). Frozen tissue pieces were pooled, weighed and chopped into small pieces on dry ice, before adding Lysis Reagent (10x Genomics, Cat# 2000558), Reducing Agent B (10x Genomics, Cat# 2000087), and Surfactant A (10x Genomics, Cat# 2000559). The tissue was homogenized using a glass douncer (MilliporeSigma, Cat# D9063) and lysed for 5 min on ice. A total of 500 µL of lysate was transferred to the Nuclei Isolation Column (10x Genomics, Cat# 2000562) to remove leftover tissue. The sample was then spun at 500 xg for 3 min at 4ºC, and the nuclei pellet was resuspended in Nuclei Separation buffer composed of Nuclei Extraction buffer (Miltenyi Biotec, Cat# 130-128-024), PBS, 10% BSA and RNase Inhibitor (MilliporeSigma, Cat# 3335402001). Miltenyi anti-nucleus beads (Cat# 130-132-997) were then added to the sample (50 µL per million nuclei) and incubated for 15 min on ice followed by an additional 2 mL of the Nuclei Separation buffer. Then the sample was purified using an LS column (Miltenyi Biotec, Cat#130-042-401) attached to the MACS MultiStand (Miltenyi Biotec, Cat# 130-042-303) with a QuadroMACS separator (Miltenyi Biotec, Cat# 130-042-302). The purified nuclei were spun at 500 xg for 5 min, and the nuclei pellet was washed with wash buffer comprised of PBS, 10% BSA and RNase Inhibitor. The nuclei suspension was centrifuged at 4ºC for 5 min at 500 xg again and the nuclei pellet was resuspended in wash buffer. Nuclei counting was done using a 1:1 dilution with acridine orange and propidium iodide on a haemocytometer (Thermo Fisher, Cat# 22-600-100). Following counting, the appropriate volume for each sample was calculated for a target capture of 20,000 nuclei and then the nuclei were loaded onto 10x 3' GEM X chip. cDNA libraries were prepared as outlined by the Chromium GEM-X Single Cell 3' Reagent Kits v4 user guide with modifications to the PCR cycles based on the calculated cDNA concentration. The molarity of each library was calculated based on library size as measured bioanalyzer (Agilent Technologies) and qPCR amplification data (Roche, Cat# 07960140001). Gene Expression libraries were sequenced on Illumina's NovaSeqX with the following run parameters: read 1 – 28 cycles, read 2 – 90 cycles, index 1 – 10 cycles and index 2 – 10 cycles.