Homo sapiens
Protocols: Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Clonetics (Lonza, Switzerland) as a pool of three donors. were cultured in EGM-2, endothelial growth medium (EBM-2, CC-3162, Lonza), supplemented with SingleQuot Bullet Kit (CC-4176, Lonza). Briefly, fresh cells were seeded in T25 flasks (Corning Costar, Sigma Aldrich, USA) and sub-cultured when they reached 70–80% confluence at a density of 5000/cm2 in humidified atmosphere of 5% CO2 at 37°C. The medium was changed at 48 h intervals. All cells tested were negative for mycoplasma infection. The passages of trypsinization and cell count are repeated until replicative Senescence, in this way the replication Senescence curve is obtained. Each step calculates population doubling (PD) which is given by the formula: (log10 F – log10 I)/log102 where I represents the number of plated cells and F the number of viable cells counted the following passage and cumulative population doubling (CPD) was calculated as the sum of all PD changes. When the value of I is equal to or less than the value of F, the cells are in replicative block. In the experiments carried out, replicative senescence was obtained from 18 replication cycles. After reaching confluence, low-passage control HUVECs and senescent HUVECs were stimulated with 25 mM D-glucose (high glucose, HG) or normal EGM-2 (5.5 mM glucose, CTR) for a week. RNA for transcriptomic assay was extracted with RNeasy Mini Kit by Qiagen (cat. no. 74104) following the manufacturer standard protocol. The quality of extracted material was assessed with NanoDrop spectrophotometer (Thermo Fisher Scientific, US). All RNA samples were purified using RNAClean XP beads by Beckman Coulter (cat. no. A63987) according to manufacturer's instructions. Efficiency of the purification and final quality control were performed with i) NanoDrop and ii) Qubit fluorometer (Thermo Fisher Scientific, US) using Qubit RNA Broad Range Assay Kit (cat. no. Q10210, Invitrogen). RNA was stored at -80°C until preparation of RNAseq libraries. The abundance of ribosomal RNA was removed from each sample using NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat) by New England BioLabs Inc. (cat. no. E7405) following the standard H-based workflow indicated in product manual and using as input 200ng of sample RNA. Further, RNA-seq libraries were prepared with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat. no. E7760S, New England BioLabs Inc.) according to the manufacturer's instructions. Each library was identified with unique index selected from a set of NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs, cat. no. E6440S, New England BioLabs Inc.). Quality and size of libraries (~ 300 bp) were confirmed using) Qubit fluorometer (Thermo Fisher Scientific, US) with Qubit DNA Broad Range Assay Kit (cat. no. Q32853, Invitrogen) and ii) Tape Station with D100 ScreenTape device (cat. no. 5067-5582, Agilent Technologies Inc.). Libraries were pooled and stored at -20°C until sequencing.
phenotype
Young
disease
none
organism
Homo sapiens
organism
Homo sapiens
collection date
not collected
scientific_name
Homo sapiens
common name
human
organism part
umbilical vein
developmental stage
neonate
isolate
not applicable
growth condition
normal glucose
cell_line
HUVEC cell line
cell_type
endothelial cell
geographic location (country and/or sea)
not collected