Total RNA was isolated from the cultured HUVECs from two donors A (Batch_A) and B (Batch_B) at population doubling levels 12 (PDL 12, young) and 39 (PDL 39, senescent) using TRIZOL reagent method (Invitrogen), respectively. Library was made with Illumina Truseq RNA Sample Preparation v2 Kit, then sequencing was carried out with Illumina HiSeq 2000 platform at BIOPIC at Peking University.
Sequencing reads that contained adapters or had low quality were pre‐filtered before mapping. Filtered reads were mapped to the hg19 genome using Tophat2 software (Version 2.0.11; Kim et al., 2013 (link)). Relative expression levels (reads per kilobase per million mapped reads, RPKM) were calculated by Cufflinks software (Version 2.2.1; Trapnell et al., 2010 (link)). Genes with RPKM <2 at both PDL12 and 39 were excluded in further analyses. The gene is defined as “transcriptionally changed or shifted” as its transcript is changed twofold in both Batch_A and Batch_B.
The senescence‐altered genes were subjected to enrichment analyses at Gene Ontology (GO;http://david.abcc.ncifcrf.gov/ ; Huang da et al., 2009 (link)).
Sequencing reads that contained adapters or had low quality were pre‐filtered before mapping. Filtered reads were mapped to the hg19 genome using Tophat2 software (Version 2.0.11; Kim et al., 2013 (link)). Relative expression levels (reads per kilobase per million mapped reads, RPKM) were calculated by Cufflinks software (Version 2.2.1; Trapnell et al., 2010 (link)). Genes with RPKM <2 at both PDL12 and 39 were excluded in further analyses. The gene is defined as “transcriptionally changed or shifted” as its transcript is changed twofold in both Batch_A and Batch_B.
The senescence‐altered genes were subjected to enrichment analyses at Gene Ontology (GO;
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