title: Bulk RNA sequencing of endometrial organoids in the IVMC protocol
design: Bulk RNA sequencing of endometrial organoids in the IVMC protocol
library_construction_protocol: Endometrial organoids were derived from biopsies of women in the secretory phase of the cycle undergoing IVF at the Bourn Hall Fertility Clinic with approval from East of England Cambridge Central Research Ethics Committee (17/EE/01551 IRAS 225205). The transfer and use of all the endometrial tissue samples together with the established endometrial organoid cultures for this study are authorized under a Material Transfer Agreements (MTA) between the University of Cambridge and the Friedrich Miescher Institute for Biomedical Research (Agreements No. G119629 and G108313). Endometrial organoids from donors B032, B044, B050, B066, B078 and B080 were used for this experiment.  (Pre-diff). Samples were collected at various timepoints across the IVMC protocol (Pre-diff, P4-diff, Horm Withdr 24h, Horm Withdr 48h, Repair 24h, E2-diff 24h, E2-diff 48h) including control conditions. Organoids were collected and Matrigel was removed using Cell Recovery Solution (Corning, 354253) for 50 min maximum on ice. The organoids were then washed with cold PBS PH (7.4) (GIBCO, 10010-015) and pellets were stored at -80oC.  Equal numbers of single cells from endometrial organoids were used as the starting material for this experiment. Endometrial organoids were collected and Matrigel was removed using Cell Recovery Solution (Corning, 354253) for 50 min maximum on ice. They were then washed with cold PBS pH (7.4) (GIBCO, 10010-015) and mechanically broken by automatic (400 times) and manual (100 times) pipetting. Organoids were, first incubated with pre-warmed accutase cell detachment solution (Gibco, A11105-01) for 5 minutes at 37oC and then, incubated with collagenase V (Sigma-Aldrich, C-9263) diluted in 10% FBS/Advanced DMEM/F12 for 20 minutes at 37oC. The digest was passed through a 40 μm nylon mesh cell strainer to ensure single cell suspension, and the flow through was collected in falcon round bottom tubes (Corning, 352235). Cell pellets were diluted in EOM, and cells were counted using the NucleoCounter® NC-202™ Automated Cell Counter (Chemometec, NC-200). Organoids growing from single cells were supplemented with 10µM Y-27632 until the formation of cystic structures. Endometrial organoids were grown at 37°C in a humidified atmosphere of 5% CO2. They were cultured in Matrigel (Corning, 356231) droplets supplemented with endometrial organoid medium (EOM).  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 ice cold Matrigel droplets, allowed to solidify at 37°C for 15-30 minutes, and covered with EOM.  Endometrial organoids were subjected to the IVMC protocol. First, they dissociated into single cells which were cultured until organoid formation (pre-diff). To mimic the proliferative phase, organoids were then primed with 500 nM β-estradiol (E2) for 48 h followed by treatment with 80 μM progesterone, 100 μg/ml cAMP and 100 mg/ml prolactin in the presence of 500 nM β-estradiol for four additional days to simulate the secretory phase (P4-diff). Hormonal treatment was then withdrawn for 48h (Horm withdr 24h, Horm withdr 48h), and then organoids were subjected to mechanical breakdown with automatic (300 times) and manual (100 times) pipetting. Organoid fragments were then replated and maintained without hormones for 24h to simulate the repair phase Repair 24h). Finally, organoids were re-exposed to E2 for another 48h to simulate the transition back to the proliferative phase (E2 diff 24h, E2-diff 48h). Controls for each timepoint included: (i) EOs subjected to hormonal treatments without breakdown (NB _H); (ii) EOs not treated with hormones but subjected to breakdown (B_NH); or (iii) EOs not subjected to breakdown (NB_NH).  Total RNA was extracted from organoid pellets using the RNeasy Micro kit with DNase treatment (Qiagen, 76004), following manufacturer's instructions.  Purified total RNA samples (150ng ng) were processed using the Illumina Stranded mRNA Prep workflow, according to the manufacturer protocol. Following library amplification (12 cycles). Library concentrations were estimated using Qubit ds DNA High Sensitivity assay kit and size distribution was assessed using D1000 Screen Tape on Agilent 4200 Tapestation  system.