Total RNA was prepared from the 3D lrECM HNSCC cell models using the RNeasy Kit with on-column genomic DNA digestion (Qiagen) as previously described [Broghammer, F. et al. Resistance of HNSCC cell models to pan-FGFR inhibition depends on the EMT phenotype associating with clinical outcome. Mol Cancer 23, 39 (2024).]. RNA integrity was assessed, and only RNA samples with RIN >= 9 were used for sequencing. mRNA was isolated from 500 ng total RNA using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat; New England Biolabs, NEB #E7400X) according to the manufacturer's instructions, with final elution in 5 µL nuclease-free water. Strand-specific RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep assay (NEB #E7760L). After ligation and user digest, adaptors were depleted using XP bead purification (Beckman Coulter) at a 0.9:1 bead-to-sample ratio. Unique dual indexing was performed during PCR enrichment (12 cycles) using amplification primers carrying the same sequence for i7 and i5 indices. After two additional XP bead purifications (0.9:1), libraries were quantified using the Fragment Analyzer (Agilent), pooled equimolarly, and sequenced on an Illumina NovaSeq 6000 in 100 bp paired-end mode, generating approximately 45 million read pairs per library on average.