RNAseq from Apc?/?Smad4?/? and their controls Apc?/?Smad4+/+ intestinal adenoma organoids in response to TGF-b1

Early passage adenomas seeded at 9,000 cells per 25µl well were used for RNA-Seq. Adenomas were grown in ADF2 medium supplemented with 8.1nM EGF, 10µM Y-27632 and 1µM SB-505142. The medium was changed on D2, the cells were washed, EGF and 390pM TGF-ß1 added and samples extracted from three different time points (t=0, t=1, t=12 hours post TGF-ß1 treatment). Three biological replicates from each genotype and RNA was pooled from 8 wells per treatment. RNA extraction was performed using Quick-RNA MiniPrep (Zymo Research, R1054), assessed using a Nanodrop-1000 spectrophotometer and RNA Clean & concentratorTM-5 (Zymo Research, R1015). RNA-Seq were performed at the Wellcome Trust Center for Human Genetics, University of Oxford. Briefly, directional multiplexed mRNA Libraries were prepared. All mRNA was ribo-depleted and converted to cDNA. Paired-end sequencing (100nt) were run on a HiSeq 2500 (Illumina) for mRNA-Seq. We used 3 biological replicates per genotype. Each replicate was sequenced on 5 lanes.