Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing
NGS (paired-end RNA-seq) of mouse hematopoietic stem and progenitor cells from young (4-6 mo) and old (31-33 mo) mice. Overall design: Wild-type HSPCs were isolated from the hind limb long bones of 4- to 6-month-old and 31- to 33-month-old male mice. c-Kit+ cells were stained and magnetically isolated from the lineage-depleted cell suspension using the EasySep mouse CD117 (c-Kit) positive selection kit (Stem Cell Technologies), following manufacturer's instructions. After overnight growth in serum-free medium (StemSpan SFEM, Stem Cell Technologies), supplemented with murine TPO (20 ng/ml, PeproTech), SCF (10 ng/ml, PeproTech), and the beta-Catenin agonist CHIR99021 (250 nM, Stemgent), HSPCs were harvested as cell pellets. Immediately after, RNA extraction was carried out with the RNeasy Plus Mini kit with genomic DNA Eliminator columns (Qiagen) in combination with on-column DNaseI digestion (Qiagen), according to the vendor's protocol. Biological duplicates of two young and two old sets of hematopoietic stem and progenitor sets were used.