Highly efficient synchronization of sheep skin fibroblasts at G2/M phase and isolation of sheep Y chromosomes by flow cytometric sorting

Abstract

that with whole genome sequencing, enrichment of Y chromosomes before sequencing might simplify data analysis and reduce sequencing costs. Flow cytometric sorting is attractive because of its capacity to purify single chromosomes in large numbers. Moreover, in recent years, sequencing technology has advanced to permit the sequencing of single chromosomes of interest isolated by flow cytometric sorting. This method has been successfully applied in humans 5,6 , gorillas 1 , Chinese hamsters 7 , and some plant species 8-11. To date, this application has not been reported with domestic animals. The Y chromosomes of many domestic animals, including the sheep Y chromosome, have not been fully assembled and characterized. In this study, we isolated and enriched sheep Y chromosomes by flow cytometric sorting and performed NGS of sorted Y chromosomes, which laid a foundation for the sequencing, assembly, and analysis of the sheep Y chromosome. Chromosomes with different sizes and AT/GC contents can be separated by flow cytometric sorting after staining with two specific fluorescent dyes (Hoechst 33258-AT specific binding and chromomycin A3-GC specific binding) 12,13. However, chromosomes with small differences in size and GC content are not easily segregated. The so-called bivariate flow karyotype is generated based on the fluorescence intensity of the two dyes, and the purity of flow-sorted chromosomes can be greater than 95% with high-quality samples and favorable conditions 12,14. Further, the flow cytometric sorting of chromosomes has been applied to DNA hybridization, DNA libraries, physical mapping, and chromosome sequencing 12. The quality of chromosome samples is critical in chromosome flow cytometric sorting 15,16. A high-quality chromosome sample contains a large number of single chromosomes in a suitable chromosome isolation buffer. For mammals, chromosome samples are usually prepared from metaphase cells. To obtain a large number of metaphase cells in a short time, cells need to be synchronized in the G2/M phase of the cell cycle. Although the preparation of chromosome suspensions from mammalian cell lines 17,18 , primary cells 19,20 , and peripheral blood lymphocytes 21,22 has been reported, conditions for cell-cycle synchronization differ among species, and this is especially the case when primary cells differ. Just like most of mammalian species, the sequences of the ovine-Y chromosome are not available. In this study, we established a highly efficient synchronization method for G2/M phase of sheep fibroblasts suitable for sheep chromosomes flow sorting. We successfully isolated and enriched ovine Y chromosomes by flow cytometric sorting and performed NGS. The purity of sorted sheep Y chromosome was analyzed using NGS data for the first time. This study demonstrates the potential application of flow cytometry for genome and chromosome research and lays a foundation for future sequencing and annotation of the ovine Y chromosome. Results Establishment, purification, and culture of sheep primary skin fibroblas...

Equal distribution of lipid droplets in daughter cells is regulated by microtubules

“…Only approximately 5% of cells in normal culture conditions are in the division phase when observed at any given time [ 17 , 18 ]. To improve the statistical efficiency of the observations, we used a double thymidine blocking method to synchronize HeLa cells ( Figure 1c ) [ 19 ]. The number of intracellular LDs was also increased to further improve observation efficiency by treating the cells with oleic acid 24 h before fixation [ 20 ].…”

Analysis of human chromosomes by imaging flow cytometry

“…Flow sorting of chromosomes has been utilized in the preparation of genomic libraries, chromosome paints, mapping of genetic markers and was crucial in the early stages of the “Human Genome Project” (Ibrahim & Van Den Engh, 2004; Monard, 2016; Van Dilla et al, 1986; Van Dilla & Deaven, 1990). In addition to application in human disease research, flow karyotyping and sorting has been crucial to isolate and understand the chromosomes of plants and animal (Doležel et al, 2021; Yao et al, 2020). Yao et al, 2020 have shown the effectiveness of flow karyotyping and sorting in isolating complex chromosomes of sheep prior to sequencing.…”

“…In addition to application in human disease research, flow karyotyping and sorting has been crucial to isolate and understand the chromosomes of plants and animal (Doležel et al, 2021; Yao et al, 2020). Yao et al, 2020 have shown the effectiveness of flow karyotyping and sorting in isolating complex chromosomes of sheep prior to sequencing. This approach has then been applied to study chromatin conformation, BAC library construction, optical mapping and sequencing amplified chromosomal DNA of plants (Doležel et al, 2021).…”

PRAMEY: A Bovid-Specific Y-Chromosome Multicopy Gene Is Highly Related to Postnatal Testicular Growth in Hu Sheep

“…To date, no study has been conducted to characterize the molecular structure and function of the ovine PRAMEY [ 24 ]. This study aimed to clone the full-length cDNA of ovine PRAMEY and determine its expression patterns, as well as to investigate its CNV in different sheep breeds and the association of CNV with testicular size in Hu sheep.…”