RNA Extraction and RT-qPCR Analysis Protocol

RNA was extracted from CRC cell lines and xenograft tumor tissues using the miRNeasy Mini Kit (Qiagen, Germantown, MD) following the manufacturer’s instructions. In brief, 3 mm cube xenograft tumor tissues were homogenized using TissueLyser II (Qiagen) with 5 mm stainless steel beads. RNA was isolated using Qiacube (Qiagen) and eluted in 50 µl of RNase-free water. Organoids were harvested in RLT lysis buffer and RNA was isolated with the RNeasy Mini Kit (Qiagen) using Qiacube and eluted in 50 µl of RNase-free water. Extracted RNA was used as a template for cDNA production using High Capacity cDNA Revers Transcription Kit (Thermo Fisher Scientific) according to manufacturer’s protocol. RT-qPCR was performed using SensiFAST SYBR mix (Bioline, London, UK) on Quant-Studio 7 PCR machine (Applied Biosystems). For specific primer sequences refer to Supplementary Table 1. All RT-qPCR target genes were calculated using ΔΔCt method normalized to β-actin. For miRNA expression analysis, we used the TaqMan real-time PCR assay kit (Applied Biosystems, Foster City, CA) and TaqMan microRNA Reverse Transcription Kit (Applied Biosystems) as described previously^{48 (link)}. For all reactions, TaqMan Universal Master Mix (Applied Biosystems) was used and the analysis was carried out using Quant-Studio 7 (Applied Biosystems). All data were analyzed using ΔΔCt method and normalized to RNU6B.