RNA sequencing of parental lung cancer cells with USP18 treatment
Description
RNA-seq data from Liu et al., 2025. A549 lung cancer cells stably expressing USP18 or empty vector treated with vehicle as a control or β-Lapachonefor 48 hours were harvested to extract total RNA for sequencing. RNA sequencing libraries were prepared using the TruSeq Stranded mRNA ligation protocol and sequenced on an Illumina NextSeq 2000 platform. Gene expression profiling from the RNA-seq data revealed differentially expressed genes and pathways following β-lapachone treatment in parental lung cancer cells, with or without gain of USP18 expression.
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Steps to reproduce
For the mRNA library preparation, we used the Illumina TruSeq stranded mRNA ligation kit. We use between 100ng to 1ug of total RNA as the input to an mRNA capture with oligo-dT coated magnetic beads. The mRNA is fragmented, and then a random-primed cDNA synthesis is performed. The resulting double-strand cDNA is used as the input to a standard Illumina library prep with end-repair, adapter ligation and PCR amplification being performed to generate the sequencing ready library. The libraries were pooled and sequenced on Illumina NextSeq2000 platform P3 flowcell using pair-end sequencing (2x101bp read length). Illumina bcl2fastq (version 2.20) was used to demultiplex and convert binary base calls and qualities to fastq format. The sequencing reads were trimmed adapters and low-quality bases using Cutadapt (version 1.18). The trimmed reads were mapped to human reference genome (hg38) and GENCODE annotation v30. The mapping was using STAR aligner (version 2.7.0f) with two-pass alignment option. RSEM (version 1.3.3) was used for gene and transcript quantification.
Institutions
- Frederick National Laboratory for Cancer Research