GSM2055960: Young; Homo sapiens; RNA-Seq
Trizol (Invitrogen) was used to extract total RNA from proliferating and senescent cells according to the manufacturer's protocol. Total RNA was used for gene expression analysis by reverse transcription (RT) followed by quantitative (q)PCR analysis or by RNA deep sequencing (RNA-Seq). RT was performed using random hexamers and reverse transcriptase (Maxima Reverse Transcriptase; Fermentas, Pittsburgh, PA, USA), and qPCR was carried out using gene-specific primers and SYBR green master mix (Kapa Biosystems, Woburn, MA, USA) in an Applied Biosystems 7300 instrument (Applied Biosystems, Life Technologies, Grand Island, NY, USA). 100 ng of RNA was used for first-strand and second-strand cDNA synthesis followed by single-primer isothermal amplification using NuGEN Ovation RNA-Seq System V2 kits according to the manufacturer's protocol. The kit amplified both polyA-tailed and non-polyA-tailed RNA and removed ribosomal RNA. The amplified cDNA was sheared using Bioruptor (Diagenode, Denville, NJ, USA) to an average size of 250–450 bases. The sequencing library was prepared using Illumina kits according to the manufacturer's protocol (Illumina, San Diego, CA, USA).
GEO Accession
GSM2055960