GSM5387184: Cnp_ko_2_snRNA; Mus musculus; RNA-Seq
Frozen tissue was transferred into 2ml of pre-chilled homogenization buffer (320mM sucrose, 0.1% NP40, 0.1mM EDTA, 5mM CaCl2, 3mM Mg(Ac)2, 10mM pH 7.8 Tris, 167uM β-mercaptoethanol and 1x protease inhibitor (Roche)). Tissue was carefully homogenized and filtered through a 80um strainer, and further centrifuged for 1min under 100 rcf. For each sample, 400 ul supernatant was collected into a pre-chilled 2ml eppendorf low-binding eppendorf tube, followed by adding 400ul 50% iodixanol solution (in 1x homogenization buffer containing 480mM sucrose) to reach a 25% iodixanol concentration. By layering 600ul of 29% iodixanol underneath the 25% iodixanol mixture, then 600ul of 35% iodixanol underneath the 29% iodixanol, two clear interfaces between different concentrations of buffers were created, and the tube was centrifuged for 20 min under 3000 rcf. After centrifuge, nuclei were collected from the band between the 29% and the 35% iodixanol layers and transferred to a fresh pre-chilled tube. The isolated nuclei were washed and resuspended in pre-chilled resuspension buffer (1xPBA, 1% BSA, 0.1U/ul RNase inhibitor). Single-nucleus RNA libraries were prepared using 10x genomics chromium single-cell 3' protocol and sequenced by Illumina Novaseq 6000