Transcriptomics of human IMR90 model of replicative senescence
Transcriptomics of human IMR90 model of replicative senescence
Human embryonic lung fibroblasts (IMR90 cells) were obtained from the Coriell institute for medical research (Camden, NJ, USA), and maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (v/v), 1 mM sodium pyruvate (GibcoTM, 11360-070, Thermo Fisher Scientific, USA) , 1x MEM non-essential amino acids (GibcoTM, 1140-035, Thermo Fisher Scientific, USA) and 1x antibiotic-antimycotic mixture (GibcoTM, 1760-250106, Thermo Fisher Scientific, USA). Cells were maintained at 37◦C in a humidified atmosphere in 5% CO2 and sub-cultured at a confluency of 80%. Cell numbers were assessed by manual counting using a Neubauer Chamber, at least in triplicates. Population doubling was tracked according to the following formula: log2(collected) - log2(seeded) Human embryonic lung fibroblasts (IMR90 cells) were obtained from the Coriell institute for medical research (Camden, NJ, USA), and maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (v/v), 1 mM sodium pyruvate (GibcoTM, 11360-070, Thermo Fisher Scientific, USA) , 1x MEM non-essential amino acids (GibcoTM, 1140-035, Thermo Fisher Scientific, USA) and 1x antibiotic-antimycotic mixture (GibcoTM, 1760-250106, Thermo Fisher Scientific, USA). Cells were maintained at 37◦C in a humidified atmosphere in 5% CO2 and sub-cultured at a confluency of 80%. Cell numbers were assessed by manual counting using a Neubauer Chamber, at least in triplicates. Population doubling was tracked according to the following formula: log2(collected) - log2(seeded) Cells were passaged at 80% confluency and harvested at the indicated population doublings. RNA was extracted using the Qiagen RNeasy Plus Mini Kit (QIAGEN, Netherlands), following the manufacturer's instructions. Residual DNA was removed using the gDNA eliminator columns from the kit. RNA concentration and quality was further evaluated using A280/260 absorbance (Nanodrop) and the Agilent RNA 6000 Nano Kit (using manufacturer's instructions, using the Bioanalyser).  NGS libraries were prepared using Illumina's Stranded mRNA Prep Ligation Kit following Stranded mRNA Prep Ligation Reference Guide (April 2021) (Document 1000000124518 v02). Libraries were prepared with a starting amount of 500ng and amplified in 10 PCR cycles. Two post PCR purification steps were performed to exclude residual primer and adapter dimers. Libraries were profiled with a DNA 1000 chip on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit 1x dsDNA HS Assay Kit, in a Qubit 4.0 Fluorometer (Invitrogen by Thermo Fisher Scientific, USA).