GSM6084094: NC10; Mus musculus; RNA-Seq
1 mL filtered tissue homogenate was then mixed with 1 mL 50% iodixanol (DKSC). The tissue mixture was added slowly into 3 mL 33 % DKSC in a 15 mL centrifuge tube for gradient centrifugation. After centrifuging at 4 °C, 3, 234 g for 20 minutes, 3.5 mL of the upper layer of the centrifuge solution was removed to a new 15 mL centrifuge tube. 1 mL cell nucleus layer at the interface of gradient centrifugation was transferred into a new 15 mL centrifuge tube, and then we added 2 mL of nuclear cleaning solution   to resuspend Buffer. Finally, we used a microscope cytometry plate stain with 0.4 % trypan blue to   detect the concentration, viability, and integrity of the nuclei. Cellular suspensions were loaded on the 10X Genomics GemCode Single-cell instrument and   performed following the manufacturer's protocol that produced single-cell Gel Bead-In-EMlusion   (GEMs). According to the manufacturer instructions, we constructed the sequencing libraries with   Chromium Next GEM Single Cell 3' Reagent Kits v3.1, which contain read sequencing primer, 10x barcode, unique molecular identifier (UMI), and poly-dT primer   sequence. Cell lysis, RNA extraction, cDNA synthesis, and amplification were automatically   completed on the Illumina sequencing platform.