GSM5901413: KO_7Years; Homo sapiens; RNA-Seq
Human samples (homo sapiens) were obtained from the Fertility Preservation Program in Pittsburgh and processed at the University of Pittsburgh. Testes were collected from 1-, 2- and 7-year-old males through the University of Pittsburgh Health Sciences Tissue Bank and Center for Organ recovery under the University of Pittsburgh CORID 686. Human samples were enzymatically digested and processed as previously described (Shami et al. Dev Cell . 2020 Aug 24;54(4):529-547.e12). Human samples were processed with approval from the University of Pittsburgh Institutional Review Board (Protocol #STUDY09020220). Isolated primary human testicular cells were processed using Chromium TM Single Cell 3′ v3 Chemistry library prep kit according to the manufacturer's instructions (10X Genomics, San Francisco, CA, USA). Isolated primary human testicular cells were processed using Chromium TM Single Cell 3′ v3 Chemistry library prep kit according to the manufacturer's instructions (10X Genomics, San Francisco, CA, USA). Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. A shallow sequencing was performed first using Illumina MiSeq SR50 to estimate the number of recovered cells in each sample (a total of 5089, 8447 and 7851 cells were collected from 1-, 2- and 7-year-old males, respectively). The sequencing of the libraries was performed a second time with a targeted sequencing depth of ~ 50,000 reads/cell using an Illumina NovaSeq 6000 sequencer (100pb single end reads) (CHGI, University of Calgary, Alberta, Canada). The raw reads were subsequently processed with a 10x Genomics CellRanger pipeline (version 3.1.0) using default settings. The outputs from the three human samples were combined using the CellRanger aggr function to normalize for the sequencing depth and produce aggregated gene-cell UMI count matrix for the downstream analysis.