GSM4567051: aged animal replicate 2; Mus musculus; RNA-Seq
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. Total RNA-seq libraries were generated and prepared for multiplexing on the Illumina platform with the TruSeq stranded total RNA library prep (Illumina) according to manufacturer's protocol. Libraries included ERCC ExFold RNA spike-in mixes (Thermo Fisher Scientific) to assess the platform dynamic range. The resultant libraries were quality-checked on a Bioanalyzer 2100 instrument (Agilent) and quantified with a Qubit fluorometer (Thermo Fisher Scientific). Paired-end
GEO Accession
GSM4567051