Bulk ATAC-seq of Foxe1KO and control Nkx2-1/mKO2+ mouse ESC-derived cells

Functional thyroid follicles generation in vitro is obtained at high efficiency by doxycycline-mediated expression of Nkx2-1 and Pax8, two proteins involved in early specification of thyroid lineage. Although those factors are sufficient to induce thyroid specification in vitro, other players, such as Foxe1, are also present in early thyroid primordium and its loss-of-function is responsible for thyroid congenital defects in mice and humans. Here we show that Foxe1KO mESCs can be induced to differentiate towards thyroid lineage but the efficiency of thyroid follicular cells generated is drastically low. Moreover, correct expression of maturation genes and capacity to produce thyroid hormone in vitro are completely disrupted. On the other hand, Nkx2-1 expressing cells derived from Foxe1KO have an unexpected ability to deviate to a lung epithelium differentiation program and form organoids containing multiple lung cell types. These findings demonstrate that Foxe1 is required to reinforce thyroid cell fate in vitro and to promote terminal maturation of thyroid follicles. Biological replicates were obtained from control and Foxe1KO cells (Nkx2-1 reporter line), at different time points of the differentiation protocol. 50000 Nkx2-1(mKO2+) cells were collected using FACS from both lines at day 10, day17 and day22. Cells derived from embryoid bodies before (day 4) and after doxycycline treatment (day7) were also collected. Samples were prepared based on Omni-ATAC protocol (Corces et al 2017). Briefly, cell pellets were resuspended in 50µl of an ice-cold cell lysis buffer (0.1% Igepal, 0.1% Tween20 and 0.01% Digitonin in Omni-ATAC Resuspension buffer). After 3 min, samples were centrifuged for 15min at 800g and subsequently, nuclei were resuspended in 50µl of reaction buffer (2.5μl Tn5 transposase, 22.5μl TD buffer, both from Nextera DNA sample preparation kit, Illumina; 16.5μl PBS, 0.5μl 1%Digitonin, 0.5μl 10% Tween20 and 5μl H20). Tagmentation reaction was performed for 30min at 37°C in a rocking plate (1000 rpm). DNA was purified using the MiniElute purification kit (QIAGEN) following the manufacturer’s indications.Barcoded libraries were PCR amplified in 11 cycles (Nextera DNA Sample Preparation Kit,Illumina). Libraries were sequenced on Ilumina HiSeq1500 system.