title: Bulk RNA-seq of co-cultured SCC25 and mouse or naked mole-rat fibroblasts
library_construction_protocol: For two-dimensional co-culture, SCC25-tdTomato cells were seeded in a 1:1 ratio with mouse or naked mole-rat fibroblasts in DMEM/F12 medium (11320-032, Life Technologies) supplemented with 0.05% FBS (Life Technologies), 400 ng/ml hydrocortisone (Santa Cruz Biotechnology) and 1% Penicillin-Streptomycin (PEST) and cultured in a 37°C incubator with 5% CO2. After 48 hours SCC25-tdTomato and fibroblasts were separated using FACS sorting (E-cadherin-APC staining to separate SCC25 from fibroblasts (TdTom &E-cadherin-). Approximately 50,000-100,000 cells were sorted of each cell type from each condition. Total RNA was extracted by adding 800 µl TRIzol reagent (Life Technologies). Total RNA samples were DNased treated using Turbo DNase (Life Technologies). RNA-sequencing libraries prepared using Illumina stranded total RNA Prep, Ligation with RiboZero Plus (Illimuna) following manufacturer's instructions.