Transcriptomic analysis was conducted on Siha cells with the HPV 16E7 gene knocked down.
RNA sequencing (RNA-seq) analysis demonstrated that the knockdown of HPV 16E7 in SiHa cells resulted in alterations in gene expression compared to the control group. Overall design: Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The quantity and purity of the total RNA were analyzed using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA), with an RNA Integrity Number (RIN) greater than 7.0. Approximately 10 µg of total RNA representing a specific adipose type was used to isolate poly(A) mRNA with poly-T oligo-attached magnetic beads (Invitrogen). Following purification, both poly(A)- and poly(A)+ RNA fractions were fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were then reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, USA). The average insert size for the paired-end libraries was 300 bp (50 bp). We then performed paired-end sequencing on an Illumina NovaSeq 6000 at lc-bio (China) following the vendor's recommended protocol.