Hiseqtm 2500 system

A Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from damaged SC samples according to the manufacturer’s instructions. Using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), the RNA quality was evaluated and verified using RNase-free agarose gel electrophoresis. Total RNA was sequenced using a HiSeqTM 2500 system (Illumina, San Diego, CA, USA) by Gene Denovo Biotechnology Co. (Guangzhou, China). RNA extraction, library construction and sequencing, Unigene expression analysis, basic unigene annotation, trend analysis, Gene Ontology (GO) enrichment analysis, and pathway enrichment analysis were performed. The unigene sequences were subjected to BLAST search against the Nr database, and the sequence that best aligned with each unigene was defined as the homologous sequence. The species to which the homologous sequence belonged was determined, and the number of homologous sequences in each species was calculated. If there was more than one homologous sequence, the first one was selected for further analysis. Next, we mapped all of the unigenes to the KOG database based on the annotation derived from the Nr protein database to predict potential gene functions and expression levels, as well as to clarify the macro-level characteristics of a species’ gene function distribution. Detailed methods are shown in Additional file 1.

HCT116 cells were transfected with pCDNA3.1-PADI3-RFP plasmids, and cells transfected with pCDNA3.1-RFP plasmids were used as controls. After 60 h of culture, the cells were harvested, and total RNA was extracted using a mirVana^TM miRNA Isolation Kit (Thermo Fisher Scientific, USA). The quality of the total RNA was evaluated using an Agilent 2100 Bioanalyzer (Agilent, USA), and RNA-sequencing was performed using an Illumina HiSeqTM 2500 system. Three independent experiments were performed to verify the results. Genes showing a ≥ 3-fold change in their expression level were selected for further study.

The RNA-sequencing experiments were performed by OE Biotech Co., Ltd. (Shanghai, China) using an Illumina HiseqTM 2500 system (Illumina, Inc., San Diego, CA, USA). RNA isolated from blood monocytes taken from young or old mice was used for RNA-seq. These RNA samples were converted into a library of cDNA fragments, Illumina sequencing adapters were added, and 50 bp single end read sequences were obtained using the Illumina HiSeq system. A quality check was performed on these sequence reads using FastQC. EdgeR software was used to analyze the data, and P < 0.05 was considered to indicate a statistically significant difference.