GSM8779823: Mm_thymus_Foxn1het_3; Mus musculus; RNA-Seq
Dissected tissues were transferred into a 1.5 ml Eppendorf tube containing 150 µl of freshly prepared ice-cold lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630, 0.1% Tween-20, 1% BSA), supplemented with 1 U/µl of Protector RNase inhibitors (Roche, 3335402001). The tissues were homogenized by pressing down with a circular motion using an RNAse-free disposable pestle for 1.5 ml tubes (Fisher Scientific, 12141364) until the majority of clumps were homogenized. Nuclei quality was assessed under a phase-contrast microscope. Subsequently, 500 µl of ice-cold wash buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA) with 0.2 U/µl of Protector RNase inhibitors was added, mixed by inversion, and spun quicky to collect liquid from the cap of the tube. The nuclei suspension was then filtered through a 70 µm Flowmi Cell Strainer (H13680-0070, Bel-Art) and centrifuged at 300g for 5 minutes at 4°C. The supernatant was removed, and nuclei pellet was resuspended in 200 µl of resuspension buffer (0.5% BSA in PBS, supplemented with 0.2 U/µl of Protector RNase inhibitors) and filtered again through a 40 µm Flowmi Cell Strainer (H13680-0040, Bel-Art). Nuclei were quantified by Trypan blue staining using the Countess 3 automated cell counter (Thermo Fisher Scientific). To exclude fat droplets form the count, only the Trypan blue-positive nuclei were counted. Each nuclei suspension was normalized to a concentration of 800 nuclei/µl using resuspension buffer. The Chromium GEM-X Single Cell 3' v4 protocol (CG000731, Rev B) was followed starting from step 1.1 according to the manufacturer's guidelines. The Chromium GEM-X Single Cell 3' Kit v4 (PN-1000686) and the Chromium GEM-X Single Cell 3' Chip Kit v4 (PN-1000690) were used to process each sample. A total of 36.6 µl of normalized nuclei suspension was added to the master mix to target a recovery of 20,000 nuclei. The ready GEM-X chip was loaded onto a 10x Genomics Chromium Xo instrument, and final libraries were completed as per 10x Genomics guidelines.