GSM9369504: 26420X2 SPiDER negative gate; Mus musculus; RNA-Seq
Single-cell RNA sequencing was performed on senescent and non-senescent cells isolated by fluorescence-activated cell sorting (FACS) using the Cytek Aurora Spectral Cell Sorter. Gastrocnemius muscles from both hindlimbs of Old mice at RL14,(n = 10 mice per group) were pooled, dissected, and digested. Cell pellets were incubated with the Senescence Detection Kit (Dojindo, SG04-01). To improve sorting , 7-AAD (1:200; BioLegend, 420404) and Hoechst 33342 (1:40,000; TOCRIS, 5117) were added to exclude debris and enrich for nucleated cells, as recommended by 10x Genomics technical support. Cells were sorted into SPiDER-βGal⁺ (senescent) and SPiDER-βGal⁻ (non-senescent) fractions. 10x Genomics Chromium GEM-X Single Cell 3' v4 Gene Expression Library Preparation NovaSeq X Series 10B Reagent Kit 150x150 bp Sequencing:  Sequencing libraries were chemically denatured in preparation for sequencing.  Following transfer of the denatured samples to an Illumina NovaSeq X instrument, a 151 x 151 cycle paired end sequence run was performed using a NovaSeq X Series 10B Reagent Kit (20085594)