Nanodrop one one microvolume uv vis spectrophotometer

For each experimental condition, cell lysates were collected using the Aurum Total RNA Mini Kit. Total RNA was isolated from duplicate or triplicate samples, and concentrations were measured using the NanoDrop One/One Microvolume UV-Vis Spectrophotometer (Thermo Fisher). CDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) and made using an Eppendorf 5331 MasterCycler Gradient Thermal Cycler (Eppendorf) with 5 ng of RNA for each planned qRT-PCR reaction. Each sample was plated in triplicate in a reaction volume consisting of 10 μl per well. Each well had 5 μl of iTaq^TM Universal SYBR^® Green Supermix, and forward and reverse primers at a concentration of 300 nM. The qRT-PCR reactions were run for 40 cycles in a Bio-Rad C1000 Touch Thermal Cycler. Gene expression analysis was conducted utilizing the delta-delta-Ct method, with GAPDH/B-actin used as normalization housekeeping gene(s). Primer sequences are reported in the Supplementary Information.

RNA was prepared from 3D tissues using QIAzol Lysis reagent and the RNeasy kit (Qiagen) according to the manufacturer's instructions. RNA concentration was determined using a Nanodrop One/One Microvolume UV–Vis Spectrophotometer (Thermofisher). cDNA was synthesised from total RNA (500 ng) using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR with DNase, according to the manufacturer's instructions.

Total RNA was extracted from the spheroids, isolated cells, human tissue, and zebrafish xenograft samples using Trizol reagent according to the manufacturer’s instructions. 100 spheroids per condition were collected in an eppendorf, tube, gently washed with PBS, disintegrated using 200μl of 0.25% trypsin-EDTA solution for 15 min at 37°C, and centrifuged at 1200 rpm for 5 min at 4°C to proceed further for RNA isolation. RNA quality was measured using NanoDrop One/One Microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific, USA) and treated with DNase I. For mRNA expression analysis, cDNA was prepared from 500ng of total RNA using the iScript cDNA synthesis kit, following the manufacturer’s guidelines. Single-cell RNA isolation was performed using a single-cell lysis kit following the manufacturer’s protocol. 100ng of total RNA was reverse transcribed using SuperScript VILO cDNA synthesis kit. PowerUp SYBR^® Green Master Mix qPCR (2X) Universal was used to perform RT-qPCR analysis in a Quant-Studio 5 Real-Time PCR System (Applied Biosystem, USA) to quantify the relative mRNA expression level using gene-specific primers. After the final extension, a melt curve analysis was performed to ensure the specificity of the products. Data were normalized to the expression of the GAPDH reference gene. Primer sequences used for RT-qPCR are listed in Supplementary Table 3.