GSM8650482: VLGN, KA; Mus musculus; RNA-Seq
Nuclei extraction from snap-frozen vLGN samples was performed according to the published literature. Using a Dounce homogenizer (D9063, Sigma), the frozen tissue (0.5 g) was homogenized in freshly made lysis buffer (500 µl, 250 mM sucrose, 0.1% Triton X-100, 0.2 U µl−1 RNase inhibitor) on ice. Part of the homogenate (200 µl) was saved for bulk RNA extraction (Qiagen RNeasy kit) and stored at −80 °C. The remaining homogenate was filtered through a 40-µm cell strainer for nuclei extraction with 4 ml of low-sucrose buffer (0.64 M sucrose, 10 mM HEPES, pH 8.0, 5 mM CaCl2, 3 mM MgAc, 0.1 mM EDTA, 0.1 mM dithiothreitol). Sonication of the homogenate was performed for 10 s. To remove cellular and myelin debris, a sucrose solution (2 M sucrose, 10 mM Tris-HCl, pH 8.0, 3 mM MgAc) was added for density gradient centrifugation at 3,200g for 30 min, and the supernatant was discarded. Nuclei were resuspended in 1 ml cold resuspension solution (1× PBS, 1% BSA, 0.2 U µl−1 RNase inhibitor) and centrifuged for 12 min at 3,200g. Nuclei were again resuspended in 50 µl cold resuspension solution. Nuclei quality and number were assessed by trypan blue staining using a hemocytometer. A yield of 1,000 intact nuclei per microliter of sample was considered to meet the criteria for snRNA-seq. Briefly, after nuclei extraction, 20,000 nuclei per sample were run on a 10x Chromium system (10x Genomics) processed for single-nucleus cDNA library preparation (Chromium Single Cell 3′ version 3, 10x Genomics) following the recommended protocol for the Chromium Single Cell 30 Reagent Kit (v3 Chemistry). Libraries were run on the NovaSeq 6000 for Illumina sequencing. All samples were judged suitable for deep sequencing. Between 33,000 and 106,000 reads per nucleus were sequenced using the Illumina HiSeq platform.