title: Illumina NovaSeq 6000 paired end sequencing; RNA-Seq of in vitro thyroid mouse organoids exposed to a dose range of phthalates for 24 hours. ATAC-Seq of human epithelial thryoid cell line Nthy-ori 3-1 exposed to DEHP or DINP (1 uM) for 5 days
design: RNA-Seq of in vitro thyroid mouse organoids exposed to a dose range of phthalates for 24 hours. ATAC-Seq of human epithelial thryoid cell line Nthy-ori 3-1 exposed to DEHP or DINP (1 uM) for 5 days
library_construction_protocol: The human epithelial thyroid cell line Nthy-ori 3-1 was plated at a density of 10,000 cells/cm2 on 6-well plates and cultured in RPMI 1640 Medium with GlutaMAX™ Supplement (61870036, Gibco), 10% FBS and Penicillin-Streptomycin (15140122, Gibco) and incubated at 37 ºC, 5% pCO2 and >95% humidity. Cells were left one day to adhere, and the following day the media was changed to culture media with DEHP (n=6) or DINP (n=6) at 1 uM in 0.5% DMSO. As solvent control, the culture media was additioned with just 0.5% DMSO (n=6). Cells were incubated for 5 days refreshing the media with the compound or DMSO only at day 3. At the end of the incubation period, cells were collected and counted manually.  50,000 cells per sample were used to prepare ATAC-Seq libraries. Libraries were prepared following the Omni-Atac protocol of Corces et al. 2017 [45] with the replacement of NP40 from the original protocol with IGEPAL (Merck, I8896-50ml). The tagmentase kit used was Illumina Tagment DNA Enzyme and Buffer Small Kit (Illumina, 20034197) and the indexes IDT® for Illumina® DNA/RNA UD Indexes Set A, Tagmentation (96 Indexes, 96 samples) (Illumina, 20027213). Seven PCR cycles were used for all samples. ATAC-Seq libraries were quantified on a Qubit 2.0 Fluorometer (ThermoFisher) and quality control checked on the 2200 TapeStation System (Agilent) or BioAnalyzer 2100 expert (Agilent).