GSM8885666: DYRK1A; Homo sapiens; RNA-Seq
Organoids were dissociated and fixed for single-cell RNA sequencing using manufacturer protocolsa standardized protocol [ref]. Organoids were first incubated in TrypLE Express (Thermo, 12604054) at 37°C for 10 minutes, with gentle pipetting using P1000 and P200 pipettes to obtain a single-cell suspension. Cells were passed through a 40 µm FlowMi filter (Merck, BAH136800040), washed with 0.04% BSA in DPBS, and centrifuged at 200×g for 5 minutes using a swinging-bucket rotor. The cell pellet was resuspended in fixation buffer (4% paraformaldehyde final concentration) and incubated overnight at 4°C. After fixation, cells were quenched in ice-cold quenching buffer, and an enhancer solution was added before gradual glycerol supplementation (10% final concentration) to improve stability. Fixed cells were stored at -80°C for up to six months. Before processing, samples were thawed at RT, centrifuged at 850×g, and resuspended in a post-storage buffer containing 0.2 U/µl RNase inhibitor (Thermo, EO0382). Propidium iodide (Miltenyi Biotec, 130-093-233) staining was used to assess cell viability, followed by fluorescence microscopy for quality control. The prepared single-cell suspensions were immediately processed on the 10x Chromium platform for single-cell RNA sequencing Barcoding and cDNA amplification were carried out using the Chromium Single Cell 3'  (vNext) Reagent Kit.