GSM8242559: 807; Mus musculus; RNA-Seq
For single nuclei dissociation, a protocol from previous study was followed [108]. Briefly, frozen tissue punches were chopped and transferred to 450ul of chilled (4°C) homogenization buffer (250mM sucrose, 25mM KCL, 5mM MgCl2, 10mM Tris buffer (pH 8.0), 1μM DTT, 1x protease inhibitor, RNaseIn 0.4 U/ul, Superasin 0.2 U/ul, 0.1% Triton X-100). After the Dounce homogenization and filtering (40μm), the nuclei were concentrated by centrifugation (1,000g for 8 min at 4°C). For staining, anti-NeuN antibody, Hoeshst, and Alexa 488 secondary antibody were treated and incubated on rocker in cold room for 30 min. The nuclei were resuspended in 600 ul FACS buffer (1% unacetylated BSA, RNaseIn 0.4 U/ul, PBS) for FACS to further purify the nuclei for sequencing. The nuclei were first sorted based on Hoechst signal and then based on Alexa 488 fluorophore, collecting 50,000 nuclei per mouse. Libraries were constructed using the 3' Expression-Single-Cell RNA-Seq using 10X Chromium System (v3.1), according to the manufacturer's instructions. The nuclei were brought to a concentration of 500 nuclei/μl and 5,000 nuclei were loaded to prepare libraries. Libraries were then sequenced (pair-end sequencing) on the Illumina NovaSeq6000 at the Iowa Institute of Human Genetics at University of Iowa. The depth of sequencing per flow cell was 50,000 reads per nuclei.