GSM3132032: T_Thy_1; Mus musculus; RNA-Seq
For thymic epithelium, thymi were isolated, cleaned of fat and transferred to DMEM + 2% FBS on ice. Thymi were minced with a razor blade and tissue pieces were moved with a glass Pasteur pipette to 15 ml tubes and vortexed briefly in 10 ml of media. Fragments were allowed to settle before removing the media and replacing it with 4 ml of digestion media containing 2% FBS, 100 μg mL-1 DNase I (Roche), and 100 μg mL-1 Liberase TM (Sigma-Aldrich) in DMEM. Tubes were moved to a 37° C water bath and fragments were triturated through a glass Pasteur pipette at 0 min and 6 min to mechanically aid digestion. At 12 min, tubes were spun briefly to pellet undigested fragments and the supernatant was moved to 20 ml of 0.5% BSA (Sigma-Aldrich), 2 mM EDTA (TekNova), in PBS (MACS buffer) on ice to stop the enzymatic digestion. This was repeated twice for a total of three 12 min digestion cycles, or until there were no remaining tissue fragments. The single cell suspension was then pelleted and washed once in MACS Buffer. Density-gradient centrifugation using a three-layer Percoll gradient (GE Healthcare) with specific gravities of 1.115, 1.065, and 1.0 was used to enrich for stromal cells. Cells isolated from the Percoll- light fraction, between the 1.065 and 1.0 layers, were then washed and resuspended for staining. Cells were sorted directly into lysis buffer from the Dynabead mRNA Direct Purification Kit (ThermoFisher). Sorted cells were pooled for bulk sequencing and poly A RNA was isolated according to the manufacturer's protocol. amplification protocol: Amplified cDNA was prepared using the NuGen Ovation RNA-Seq system V2 kit, according to the manufacturer's protocol Sequencing libraries were generated using the Nextera XT library preparation kit with multiplexing primers, according to manufacturer's protocol (Illumina)