title: Illumina HiSeq 4000 sequencing; Single-cell RNA-seq of Endometrial organoids treated with NOTCH or WNT inhibitors in a time course of Estrogen and Progesterone stimulation
design: Single-cell RNA-seq of Endometrial organoids treated with NOTCH or WNT inhibitors in a time course of Estrogen and Progesterone stimulation
library_construction_protocol: 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 v2 chemistry Libraries were constructed using the 10x v2 chemistry