GSE161511
Single-cell RNA-seq analysis of regulatory role of Gm4665 in spermatogenesis
Purpose: To understand the likely mechanisms behind the effects of candidate lncRNA knockdown, we applied single-cell RNA-seq (scRNA-seq) to decipher transcriptional alterations of affected germ cells induced by Gm4665 knockdown. Methods: We removed somatic cells and isolated RFP+ germ cells infected with AAV9-RFP from shCtrl or Gm4665-depleted testes by FACS, before performing scRNA-seq with the Chromium system (10x Genomics). For scRNA-seq, the gene cell barcode matrix was filtered based on the number of genes detected per cell (any cells with fewer than 500 or more than 4000 genes per cell were filtered out) and the percentage of mitochondrial UMI counts (any cells with more than 10% of mitochondrial UMI counts were filtered out). Results: We separately analyzed the differentially expressed genes (DEG) induced by Gm4665 knockdown in rST and elST populations. This demonstrated that Gm4665 knockdown resulted in increased expression of 190 genes and decreased expression of 328 genes in rST, and increased expression of 135 genes and decreased expression of 35 genes in elST. Conclusions: Single-cell transcriptional analysis of male germ cells indicated the functional requirement of Gm4665 for multiple aspects of spermatid differentiation during spermiogenesis. Overall design: We isolated RFP+ germ cells from 3 mice per condition, including shCtrl, Gm4665-shRNA1#, and Gm4665-shRNA2#.