GSM8447101: DG_epilepsy, snRNA-Seq; Mus musculus; RNA-Seq
The tissues samples were surgically removed and snap frozen in liquid nitrogen for intact nucleus isolation. The nucleus was isolated and purified as previously described with some modifications. Briefly, the frozen tissue was homogenized in NLB buffer which contain 250 mM Sucrose, 10 mM Tris-HCl, 3 mM MgAc2, 0.1% Triton X-100 (SigmaAldrich, USA), 0.1 mM EDTA, 0.2U/μL RNase Inhibitor (Takara, Japan). Various concentration of sucrose was used to purify the nucleus. The concentration of nucleus was adjusted to about 1000 nuclei/μL for snRNA-Seq. The snRNA-Seq libraries were generated using the 10X Genomics Chromium Controller Instrument and Chromium Single Cell 3' V3.1 Reagent Kits (10X Genomics, Pleasanton, USA). Briefly, cells nuclei were concentrated to approximately 1000 nuclei/μL than loaded into each channel to generate single-cell Gel Bead-In-Emulsions (GEMs). After the RT step, GEMs were broken and barcoded-cDNA was purified and amplified. The amplified barcoded cDNA was fragmented, A-tailed, ligated with adaptors and index PCR amplified. The final libraries were quantified using the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific, USA) and the size distribution of the libraries were determined using a High Sensitivity DNA chip on a Bioanalyzer 2200 (Agilent, USA). All libraries were sequenced by Novaseq6000 (Illumina, USA) on a 150 bp paired-end run.