GSM8032544: PF_MUT_rep2; Mus musculus; RNA-Seq
The petrous bone and paraflocculus/flocculus (PF/F) were dissected from P5.5 Tbx1+/- mice and their matched littermates. Each sample was flash frozen and stored at -80°C until used. The frozen bones (5 mice per group) were placed into the prechilled tissue tubes (Covaris, cat. no. 520001) on dry ice and pulverized using a prechilled CP01 cryoPREP manual dry pulverizer (Covaris, cat. no. 500230). The coarsely powdered bone was placed into a well of 6 well plate with 1ml of CHAPS/salt-Tris solution (ST buffer) containing 980 µl of 1 % CHAPS (Millipore, cat. no. 220201) mixed with 1 ml of 2xST buffer (292 mM NaCl, 20 mM Tris-HCl pH 7.5, 2 mM CaCl2 and 42 mM MgCl2), 10 µl of 2 % BSA (New England Biolabs, cat. no. B9000S) and 10 µl of nuclease-free water, and gently chopped using Noyes spring scissors (Fine Science Tools, cat. no. 15514-12) for 2 minutes on ice. The nuclei suspension was filtered through a 40 µm strainer into a 15 ml tube (pre-blocked with 2.5% BSA). The filter was washed with 1ml of 1x ST buffer and the filtered volume was brought up to 5 ml by addition of 1x ST buffer. Following additional filtration through a 30 µm strainer, the solution was centrifuged at 300xg for 5 minutes. The pellet was resuspended in 1% BSA in PBS (250-500 µl based on pellet size) and filtered through a strainer cap FACS tube (Corning, cat. no. 352235). Nuclei were counted and checked for integrity under a microscope and the volume was adjusted to a concentration of 1x106 nuclei/ml. The frozen PF/F (5 mice/group) was placed directly into 1 ml of CHAPS/ST buffer (without pulverization) and chopped for 3 minutes on ice and processed further as described above. All solutions used for nuclei isolation were amended with 0.2U/µl of RNase Inhibitor (Roche, cat. no. 3335399001). Nuclei from developing mouse cerebellar tissue or petrous bone were isolated and processed using the 10x Chromium technology, specifically optimized for single nuclei RNA sequencing. The nuclei were processed using the Chromium Single Cell 3′ v3 or v3.1 Library & Gel Bead Kit, which is designed for nuclei and includes adjustments to the standard single-cell protocol to accommodate the unique characteristics of nuclei. All procedures were carried out in accordance with the manufacturer's recommendations for single nuclei RNA-seq snRNA-seq