A cell-autonomous human model of pathway dysfunction in lung squamous cell carcinoma development
Air liquid interface organotypic cultures were washed once with PBS, snap frozen on dry ice and stored at -80C for a maximum of 2-3 days. RNA was extracted using the RNeasy Mini kit (Qiagen, 74104) according to the manufacturers instructions. Specifically, each individual ALI culture was lysed using 200l of RLT buffer. DNase digestion was performed on-column using the On-Column DNase I Digestion Set (Sigma Aldrich, DNASE70) as instructed. Finally, RNA was eluted in 30l of RNase-free water, snap frozen on dry ice and stored at -80C. Library preparation and RNA sequencing was carried out by the CRUK Manchester Institute Molecular Biology Core Facility. RNA quality was assessed using the Agilent 2100 Bioanalyzer and samples with a RIN value of >7 were selected for sequencing. Library preparation was performed using 100ng of total RNA using the Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit. Single-end sequencing with 100bp read length was performed using a Novaseq 6000 sequencer (Illumina).