Bulk RNA-Seq of human thyroid organoids

The function of the thyroid gland is to metabolize iodide to synthesize hormones that act on almost all tissues and are essential for normal growth and metabolism. Low plasma levels of thyroid hormones lead to hypothyroidism, which is one of the most common diseases in the general population and cannot be always satisfactorily treated by lifelong hormone replacement. Therefore, in addition to the lack of in vitro tractable models to study human thyroid development, differentiation and maturation, there is a need for new therapeutic approaches that involve replacement of thyroid tissue to better control hormone balance. Here we report the first model of thyroid organoids derived from human embryonic stem cells that produce thyroid hormones in vitro and are capable of restoring plasma thyroid hormone levels when transplanted into athyreotic mice. Bulk RNA-seq was performed in NKX2-1GFP+ hESC-differentiated cells every week from day 16 to day 45 of our differentiation protocol. 10,000 NKX2-1GFP+ cells were directly sorted into 700µl of Qiazol lysis reagent (Qiagen) and RNA isolation was performed using the miRNeasy micro kit (Qiagen) according to the manufacturer's instructions. Ovarion Solo RNA-seq Systems (NuGen) was used as indicated by the manufacturer. Multiplexed libraries (10ρM) were loaded onto flow cells and sequenced on the HiSeq 1500 system (Illumina) in high-output mode using the HiSeq Cluster Kit v4 (Illumina).