NextSeq 550 paired end sequencing; snATAC-seq of the developing mouse cerebellum across eleven stages
snATAC-seq of the developing mouse cerebellum across eleven stages
The frozen tissue was homogenized on ice in 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl (pH 8), 0.1% IGEPAL, 1 μM DTT, 0.4 U/μl Murine RNase Inhibitor (New England BioLabs), 0.2 U/μl SUPERas-In (Ambion), cOmplete Protease Inhibitor Coctail (Roche) by trituration and/or by using a micropestle. After 5 minutes incubation the remaining bits of unlysed tissue were pelleted by centrifugation at 100 g for 1 minute. The cleared homogenate was centrifuged at 400 g for 4 minutes to pellet the nuclei. Nuclei were washed 1-2 times in the homogenization buffer, collected by centrifugation and resuspended in the Nuclei Buffer (10X Genomics). If needed the nuclei were strained using 40 μm Flowmi strainers (Sigma). For estimation of nuclei concentration, Hoechst DNA dye was added and nuclei were counted on Countess II FL Automated Cell Counter (Thermo Fisher Scientific). Cerebelli were dissected as whole or in 2 halves. E10.5 and E11.5 samples were pooled from 2-4 littermates. During dissection the developmental samples were kept in ice-cold PBS and most of the meninges was removed. All samples were snap-frozen in liquid nitrogen. 15,000 nuclei per sample were subjected to tagmentation, single cell barcoding and library construction using the Chromium Single Cell ATAC Reagent kits (v1) and Chromium Controller instrument (10X Genomics) according to the manufacturer's protocols. Libraries were prepared using the Chromium Single Cell ATAC Reagent kits (v1) according to the manufacturer's protocols.