Single-cell Multi-omics Atlas of Somites from Normal and Growth-Retarded Duroc x Tibetan Pig Embryos at E21

Somite tissues were isolated from Duroc×Tibetan pig (DZ) at E21 and washed with pre-cooled phosphate buffer saline (PBS) for three times. Then, cut tissues into small pieces and transferred to pre-warmed digestion medium containing 0.1g/mL collagenase I (Sigma-Aldrich, USA) and 100μg/mL DNase I (Sigma-Aldrich, USA) in RPMI 1640. The tissues were digested for 45 minutes in an incubator at 37℃, with shaking every 5 minutes to release the cells. Released cells were passed through a 70µm cell strainers (BD Biosciences, USA) and transferred to a 15ml centrifuge tube. Centrifuge at 500×g for 5 minutes at room temperature, discard the supernatant and wash once with PBS. Sample viability was assessed by Trypan Blue (Thermo Fisher, USA) and an automated cell counter (Countstar, China). Preparation of single cell water-in-oil droplets according to 10×Genomics Single Cell 3' Reagent Kits v2 Guide. Droplets were reverse transcribed and amplified by Veriti 96-well thermal cycler (Thermo Fisher, USA). cDNA was then purified using SPRIselect magnetic beads (Beckman, USA) and on a bioanalyzer to determine the concentration. According to the Single Cell 3’Reagent Kit v3 user guide to prepare cDNA libraries.The raw data gene expression matrix was processed using R (v.3.5.2). Dimensionality reduction, clustering, and visualization of scRNA-seq data were conducted using Seurat (v.4.0) with default parameters. ScATAC-seq data were analysed by ArchR. The raw data files are in FASTQ format, while the processed data include features (genes/peaks), cell barcodes, and expression/accessibility count matrices.