GSM6507307: snRNA, Cerebrum, Cdca7G305V/G305V, rep1; Mus musculus; RNA-Seq
Nuclei were isolated as previously described (Krishnaswami et al, 2016). Briefly, tissue was chopped, flash frozen and stored at -80 ºC. The Dounce homogenizer (2 ml one), pestles and all the buffers (NIM1 – 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris pH 8; NIM2 – NIM1, 1 µM DTT, protease inhibitor; Homogenization buffer – NIM2, 0,4 U/µl RiboLock (ThermoFisher, EO0384)) were precooled. The tissue was homogenized in 1 ml homogenization buffer with five strokes of the loose pestle, followed by 15 strokes of the tight pestle. Homogenate was filtered through a BD Falcon tube with a 35-μm nylon cell strainer cap (352235, Corning). The number of nuclei was counted by staining an aliquot with trypan blue and using TC10 Automated Cell Counter (BioRad). The nuclei were resuspended in 1 ml NIM2 buffer and sample was centrifugated (500 g, 5 min, 4 ºC). The washing step was repeated 2-3 times. snRNA-seq libraries were obtained using Chromium Next GEM Single Cell 5' Library & Gel Bead Kit v1.1 (10x Genomics , PN-1000165) chemistry and Chromium Next GEM Chip G Single Cell Kit (10x Genomics, PN-1000120). Two libraries were prepared per genotype. 5' snRNA-seq