GSM8012231: aortic tissue of CA1 knockin ApoE-/- mice fed high-fat, methoxazolamide treated after atherosclerosis was established; Mus musculus; RNA-Seq
The nuclear suspension preparation experiment was conducted on ice. Fresh or frozen tissue samples were chopped in 1ml of cell lysate (NST), which was divided into 0.1% NP40, 10mM Tris-HCl, 146mM NaCl, 1mM CaCl2, 21mM MgCl2 and 1U/μl RNase inhibitor. The cracking time was 7min. After the cells were completely lysed by Trypan blue microscopy, 1ml ST Wash buffer (10mM Tris-HCl, 146mM NaCl, 1mM CaCl2, 21mM MgCl2) was added. 0.01% BSA (NEB B9000S) and 40U/mL RNase inhibitor). Filtrate was transferred to a 15mL centrifuge tube through a 40µm cell sieve (BD). The filtrate was rinsed with an appropriate amount of ST Wash buffer and combined with the nuclear filtrate. Centrifuge the horizontal rotor 500g at 4℃ for 5 min. The nucleus was re-suspended with 5ml PBS+1%BSA, and after washing and centrifugation, 100µl PBS+1%BSA was re-suspended. Trypan blue staining, microscopic counting. Nuclei were diluted to 700-1200/µl with PBS+1%BSA. Machine and cDNA libraries were amplified according to the operating instructions of 10×Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (1000268). The DNA library was constructed using Chromium™ single cell 3'/5' library construction kit (1000020). The constructed library was sequenced using the Illumina Nova 6000 platform in PE150 sequencing mode.