Bulk RNA-seq of human esophageal squamous cell carcinoma cell lines: CRISPR-mediated depletion of P3H1
Ribosomal RNA (rRNA) depletion was performed using QIAseq FastSelect rRNA HMR Kit. cDNA libraries were prepared by KAPA RNA HyperPrep Kit. One hundred nanogram of total RNA was used as starting material for ribosomal RNA depletion. Cytoplasmic rRNA, mitochondrial rRNA were removed by the QIAseq FastSelect reagents during the NGS library preparation. The processed RNA was fragmented to 200  300 bp in the presence of magnesium ions. The fragmented rRNA-depleted RNA was then applied as template to synthesize the first-strand cDNA by using random hexamer-primer and reverse transcriptase. In the second strand cDNA synthesis, the mRNA template was removed and a replacement strand was generated to form the blunt-end double-stranded (ds) cDNA. The ds cDNA underwent, 3 adenylation and indexed adaptor ligation. The adaptor-ligated libraries were enriched by 15 cycles of polymerase chain reaction (PCR).The libraries were denatured and diluted to optimal concentration. Illumina NovaSeq 6000 was used for Pair-End 151bp sequencing.