Nthyori_DMSO_3_smallRNA
Homo sapiens
Protocols: The human epithelial follicular cell line Nthy-ori 3-1 was seeded at a density of 40,000 cells/cm^2 in a 6-well plate exposed for 24 hours in triplicate to 1 μM or 10 μM benzo[a]pyrene (BAP) (B-1760, Sigma) dissolved in DMSO (1029521000, Merck) (final concentration of DMSO 0.5%). Six 0.5% DMSO solvent controls were also included. The medium used for cell culture was RPMI 1640 Medium, GlutaMAX™ Supplement (61870036, Gibco) supplemented with 10% FBS and 100 U mL-1 Penicillin-Streptomycin (15140122, Gibco). Enriched thyroid follicles were exposed to DMSO 0.5% for 24 hours (5 biological replicates). The medium used was DMEM (31966021, Gibco) supplemented with 15% FBS, vitamin C (50 μg mL-1) (A4544, Sigma), nonessential amino acids (0.1 mM) (11140035, Gibco), sodium pyruvate (1 mM) (11360039, Gibco), penicillin and streptomycin (50 U mL-1) (15140122, Gibco), 2-mercaptoethanol (0.1 mM) (31350010, Gibco), 8-Br-cAMP (10 µM) and TGF-βRI inhibitor SB431542 (10 µM) (1614, Tocris). At the end of the exposure time, cells were collected in 1.5 mL Eppendorf tubes and washed once with PBS. They were then lysed in QIAzol Lysis Reagent (79306, Qiagen). Murine thyroid organoids were derived from recombinant embryonic stem cell line (A2LoxNkx2-1-Pax8). Total RNA was extracted using the Direct-zol RNA miniprep (R2051, Zymo Research) for Nthy-ori 3-1 cells. Total RNA was extracted using the miRNAeasy Micro Kit (217084, Qiagen) for the thyroid follicles. All prepared 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). 50 ng of total RNA isolated from Nthy-ori 3-1 samples were used as input for the NEXTFLEX® Combo-Seq™ mRNA/miRNA Kit (NOVA-5139-53, PerkinElmer). All RIN (RNA Integrity Number) values were 8 or higher. To deplete tRNA fragments and Y RNA fragments during the library preparation, the NEXTFLEX® tRNA/YRNA Blocker was used following the manufacturer's instruction. We performed 13 PCR cycles during library preparation. 20 ng of total RNA isolated from thyroid organoids samples were used as input for the NEXTFLEX® Combo-Seq™ mRNA/miRNA Kit (NOVA-5139-53, PerkinElmer). All RIN (RNA Integrity Number) values were 8 or higher. To deplete tRNA fragments and Y RNA fragments during the library preparation, the NEXTFLEX® tRNA/YRNA Blocker was used following the manufacturer's instruction. We performed 16 PCR cycles during library preparation. Poly(A) libraries were prepared on an automated system (Zephyr G3® NGS) with the NEXTFLEX® Rapid Directional RNA-Seq Kit 2.0 (NOVA-5198-02, PerkinElmer) using 1 μg of total RNA extracted from Nthy-ori 3-1 samples and performing 10 PCR cycles. NEXTFLEX® Poly(A) Beads 2.0 (NOVA-512992, PerkinElmer) and NEXTFLEX® Unique Dual Index Barcodes (NOVA-512923, PerkinElmer) were used during the library preparation. Small RNA libraries were prepared manually with the NEXTFLEX® Small RNA-Seq Kit v3 (NOVA-5132, PerkinElmer) from 100 ng of total input Nthy-ori 3-1 RNA and performing 18 PCR cycles.