title: Ion Torrent S5 XL sequencing; Strategic destabilization of K-MT attachments in elderly cells delays senescence
design: Strategic destabilization of K-MT attachments in elderly cells delays senescence
library_name: 87y_Kif2B_2_s
library_construction_protocol: Human dermal fibroblasts (HDFs) retrieved from skin samples of neonatal (No. DFM021711A, Zen Bio) and octogenarian (AG10884, Coriell Institute) Caucasian males were used. All donors were reported as “healthy”. For all experiments, HDFs were seeded at 1x104 cells per cm2 of growth area in minimal essential medium Eagle–Earle (MEM) supplemented with 15% fetal bovine serum (FBS), 2mM L-glutamine, and 1x antibiotic-antimycotic (all from Gibco, Thermo Fisher Scientific). Only early passage dividing fibroblasts (up to passage 3–5) with cumulative population doubling level PDL < 24 were used. Total RNA from asynchronous cell populations was extracted using RNeasy® Mini Kit (Qiagen).   10ng were reverse-transcribed using the AmpliSeq Whole Transcriptome primers with the included SuperScript VILO cDNA Synthesis kit (Thermo Fisher Scientific). cDNA was used for target amplification (12 cycles) with Ion AmpliSeq primers and technology. Barcoded adapters were added and ligated to individual reactions according to the instructions of Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific). The pooled libraries were processed on Ion Chef™ System(Thermo Fisher Scientific).
attributes.tag: Experimental Factor: cell line
attributes.value: AG10884