RNA-seq Data Generation from Cell Samples

Previously generated RNA-seq data from the same samples (4M, 6M, and 10M) were used in this study (GSE108279 [20 (link)]). Briefly, CA samples were thawed on ice and total RNA was extracted with the RNeasy Mini Kit (Qiagen, Gaithersburg, MD, USA), following the manufacturer’s recommendations. Total RNA concentration was measured using a NanoDrop DP-1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and a Bioanalyzer (Agilent, Santa Clara, CA, USA) to evaluate concentration, purity, and integrity. All samples had a 230/260 ratio > 1.8, a 260/280 ratio > 2.0, and an RNA integrity number > 8.0. Library preparation was performed using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA, USA), as per the manufacturer’s instructions. Sequencing was performed on a HiSeq 4000 (Illumina) using a HiSeq 4000 sequencing kit version 1, generating 150 bp paired-end reads. Fastq files were generated and demultiplexed using bcl2fastq v2.17.1.14 Conversion Software (Illumina).