Illumina NovaSeq 6000 sequencing; Single-cell RNA-seq of Endometrial organoids treated with NOTCH or WNT inhibitors in a time course of Estrogen and Progesterone stimulation
Single-cell RNA-seq of Endometrial organoids treated with NOTCH or WNT inhibitors in a time course of Estrogen and Progesterone stimulation
Endometrial scratch samples from secretory phase endometrium for deriving organoids  were obtained from Bourne Hall Clinic under ethical approval from the East of England-Central  Research Ethics Committee for the 'Biology of the Human Uterus in Pregnancy and Disease  Tissue Bank' run by the Centre for Trophoblast Research (17/EE/0151).  Organoids at 37°C in a humidified atmosphere of 5% CO2. The medium was refreshed every 2-3 days and the organoids were passaged at an average ratio of 1:3 every 5-7 days. The organoid suspension was centrifuged for 6 minutes at 600×g between passaging steps. The passaged organoid pellet was resuspended in 25 μl ice cold Matrigel (Corning, 356231) droplets, plated in a 48-well plate (Costar, 3548), allowed to solidify at 37°C for 15-30 minutes, and covered with 250 μl endometrial organoid expansion medium (ExM). 10,000 single cells were plated per 25 μL Matrigel droplet into a 48-well plate with ExM supplemented with Rho kinase inhibitor (Y-27632-CAS 146986-50-7) and CHIR 99021.  Ten days after plating, organoids were primed with 10 nM E2 and treated with NOTCH and WNT inhibitors (20 μM DAPT, 1 μM DBZ, 2 μM IWP-2, 2  μM XAV939 in the ExM). Rspondin-1, a WNT signalling activator, was deprived from the ExM in the conditions where WNT inhibitors were used. After 48 hours, they were stimulated with 10 nM E2, 1 μM P4, 100 μg/ml cAMP, 20 ng/ml PRL) while still being treated with NOTCH and WNT inhibitors (20 μM DAPT, 1 μM DBZ, 2 μM IWP-2, 2 μM XAV939). RNA was converted into cDNA and amplified as part of the 10x v3 chemistry Libraries were constructed using the 10x v3 chemistry