Single-cell analysis reveals gene expression profiles and cell types in wooden breast myopathy

Wooden Breast (WB) is a novel myopathy affecting the pectoralis major muscle of modern broiler chickens. We selected pectoralis fillet samples, a normal(healthy) fillet and a WB fillet. Overall design: Normal fillets and severe WB fillets were dissociated with enzymatic digestion with collagenase I for 30 minutes at a concentration of 0.15 g per 100 mL. Then single-cell suspension were obtained by passing the dissociated cell suspensions through a 40 µm cell strainer. All isolated cells were applied to magnetic bead separation method to remove dead cells. Then trypan blue (Thermo Fisher Scientific, waltham, MA, USA) and Automated Cell Counter (TC 20TM, BIO-RED Inc., CA, USA) were used to evaluate the viability of filtrated single cells. Noteworthy, the lipid cells were excluded. Following counting, the appropriate volume was calculated for a target capture of 8000 cells. Samples below the required cell concentration as defined by the user guide were pelleted by beads containing cell Barcode, and then formed as Gel Bead-in-Emulsions. Therewith, reverse transcription and RT-PCR reaction performed in those beads, and cDNA library construction were made using the 10x Genomics Single Cell v2 kit following the 10x Genomics protocol. Libraries were sequenced on the Illumina NovaSeq 6000 system.