title: Transcriptional profiling of healthy versus cystic fibrosis human endometrial organoids upon hormonal exposure
design: Transcriptional profiling of healthy versus cystic fibrosis human endometrial organoids upon hormonal exposure
library_construction_protocol: Healthy and CF endometrial organoids (EO), grown in endometrial organoid medium (EOM) without hormones or EOM supplemented with hormones (β-estradiol (E2) or combination of E2, progesterone, cyclic adenosine monophosphate and Wnt inhibitor XAV-939 (referred to as “EPCX”)). EO were harvested from Matrigel droplets using ice-cold DMEM/F12. Collected organoid samples were lysed using lysis buffer from the RNeasy Micro kit (Qiagen) following manufacturer's instructions.  Endometrial biopsies were obtained at UZ Hospitals Leuven, after informed written consent, from CF patients, and from controls, i.e. healthy volunteers or patients undergoing laparoscopy for benign non-endometrial gynecological conditions. The controls all tested negative for carrier status of F508del. The tissue samples were mechanically and enzymatically dissociated into small fragments and single cells, which were resuspended in 70% Matrigel (Corning)/30% DMEM/F12 (Thermo Fisher Scientific) supplemented with Rock inhibitor (RI, Y-27632, Merck Millipore) and seeded in droplets and cultured in endometrial organoid medium (EOM) for expansion. EO were passaged every 7–14 days, including dissociation with TrypLE Express (Thermo Fisher Scientific), containing RI and mechanical trituration, resuspended in 70% Matrigel/30% DMEM/F12 (supplemented with RI) and re-seeded. Hormone exposure to mimic the menstrual cycle phases was performed as follows: after 2 days of growth in endometrial organoid medium (EOM), the EO were exposed for 6 days to 10 nM β-estradiol (E2, Sigma), or for 2 days to 10 nM E2 followed by 4 days to a combination of 10 nM E2, 1 µM progesterone (P4, (Sigma), 0.25 mM cyclic adenosine monophosphate (cAMP, Tocris) and 10 µM Wnt inhibitor XAV-939 (Tocris) (referred to as “EPCX”). Medium was refreshed every 2 days. EO were collected at the end of hormone exposure (day 8) for RNA extraction. RNA was extracted using the RNeasy Micro kit (Qiagen). RNA concentration and purity were determined using the Nanodrop ND-8000 (Nanodrop Technologies) and RNA integrity and concentration were assessed using the BioAnalyzer RNA Nano-kit (Agilent). Per sample, an amount of 250 ng of total RNA was used as input. Using the Illumina Stranded mRNA Sample Prep Kit (protocol version: # 1000000124518 v02 (April 2021)) poly-A containing mRNA molecules were purified from the total RNA input using poly-dT oligo-attached magnetic beads. The purified mRNA was fragmented and in a reverse transcription reaction using random primers and Actinomycin D, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using dUTP to achieve strand specificity. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which pre-index anchors were ligated preparing the fragments for dual indexing. Anchor-ligated fragments were then purified using magnetic beads. Finally, enrichment PCR was carried out to enrich those DNA fragments that have anchor-ligated DNA fragments and to add indexes and primer sequences for cluster generation.