RNA-seq of 3T3-TGF+Yoda1 3
NIH/3T3 cells, a mouse fibroblast cell line, were procured from ATCC. 3T3 cells were cultured in DMEM/F-12 medium supplemented with 10 percent Serum Plus II (DF10) and maintained at 37 C in a humidified atmosphere containing 5 percent CO2. For experiments, approximately 70,000 cells per well were seeded into 12-well plates, and treatments were initiated once the cells reached 6070 percent confluence. Due to the high mortality of 3T3 cells in serum-deprived medium, all treatments were performed in 2 percent serum-supplemented medium (DF2). This group was treated with TGF-beta (10ng/ml)  and Yoda1 (10uM) for 24 hours before RNA isolation. The Yoda1 + TGF group received a 20 min pre-treatment of Yoda1 alone followed by the addition of TGF.  Samples were sequenced to an average depth of 50 million paired-end reads per sample. Stranded RNA-seq libraries were prepared using poly(A) enrichment and sequenced on an Illumina NovaSeq 6000 platform using an S4 flow cell with paired-end 100 bp reads.