CNOT1 and Transcriptomic Landscape of a HeLa Cell Line

Purpose: The goals of this study are to compare NGS-derived total transcriptome profiling (RNA-seq) of CNOT1-depleted cells (using siRNA) with control HeLa cells before and after DNA damage to examine quantitative gene expression. Methods: 72 hours post siRNA transfection control and CNOT1-depleted HeLa cells were mock-treated or treated with HU 0.25m, DRB 100µm or MMC 1µm. Cells were collected at indicated timepoints for each treatment. Total RNA was extracted from control and CNOT1-depleted HeLa cells using RNeasy Plus Mini (Qiagen). The quality and quantity of extracted RNA were measured using a Qubit 2.0 Fluorometer (Life Technologies) and an Agilent 2200 TapeStation system, respectively. Dual-indexed, strand-specific RNA-Seq library was prepared from submitted total RNA, using RiboZero (Illumina) rRNA depletion and the NEBNext Ultra Directional RNA library preparation kit (NEB). RNA-Seq was performed on 2 lanes of HiSeq4000 (paired-end, 2x150bp) platform to generate ~80M reads per sample. The raw data files were generated in binary base call (BCL) format and converted to FASTQ format using bcl2fastq Conversion Software (Illumina). FASTQ format readings were transferred to BaseSpace® Sequence Hub (Illumina). The assay was run in 4 lanes in paired-end and 76-cycle mode. The FASTQ files were aligned using HISAT2 (Kim, Langmead et al. 2015), and the resulting mRNA transcripts were assembled using StringTie (Pertea, Pertea et al. 2015) as described in (Pertea, Kim et al. 2016).