GSM5328093: spermatogonia1(SPG1); Sus scrofa; RNA-Seq
Testis cell suspensions were loaded into Chromium microfluidic chips with 3' v3 chemistry and used to generate single-cell gel bead emulsions (GEMs) using the Chromium controller (10X Genomics), GEM-RT was performed in a T100 Thermal cycler (Bio-Rad) and all subsequent steps to generate single-cell libraries were performed according to manufacturer recommendations. Fractions with high purity of spermatogonia, pachytene spermatocytes and round spermatids were resuspended with Trizol (Invitrogen, USA) and total RNA was extracted for bulk RNA-seq. RNA seq: The rRNAs were removed from total RNA by using NEBNext rRNA Depletion Kit (New England Biolabs, Inc., Massachusetts, USA) following the manufacturer's instruction. NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Inc., Massachusetts, USA) was used to construct RNA libraries. Library quality was confirmed by BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). Library sequencing was performed on an Illumina Hiseq 4000 with 150 bp paired end reads. scRNA-seq: Single-cell suspensions were loaded to 10x Chromium to capture 16,966 single cell according to the manufacturer's instructions of 10X Genomics Chromium Single-Cell 3' kit (V3). The following cDNA amplification and library construction steps were performed according to the standard protocol. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run, 150bp) by LC-Bio Technology co. ltd., (HangZhou, China) at a minimum depth of 25,000 reads per cell.