Exchange of subtelomeric regions between chromosomes 4q and 10q: a novel way to revert the FSHD genotype and phenotype
In this study we used the CRISPR-Cas9 technique to induce the targeted 4q35;10q26 translocation in FSHD myoblasts. We were able to demonstrate that t(4;10)(q35;q26) can generate recombinant genotypes which revert the pathological phenotype of the FSHD cells. Overall design: RNA-seq was performed for the control (AB1190) and FSHD (AB1080) cell lines, as well as the translocation clones with the improved phenotype (AB1080-T5, AB1080-T6). The cells were collected at the myoblasts stage and at the stage of myotubes after four full days of differentiation. For the non-differentiated samples, 5 × 10^5 cells were collected and subjected to the RNA extraction right away. For the differentiated samples,  5 × 10^5 cells were seeded to 6-well plates to obtain ~90% confluency on the next day, when the differentiation was induced by changing the proliferation medium to the differentiation medium with low serum content (98% DMEM (Sigma-Aldrich #D0822), 2% FBS (Life technology #10270), 1 % penicillin– streptomycin (Gibco #15140-122)). On the 5th day of differentiation, the myotubes were detached with Trypsin-EDTA (Sigma-Aldrich #T2601) and subjected to the RNA-extraction. Total RNA was isolated using the Nucleospin RNA isolation kit (Macherey–Nagel #740955). All samples were prepared in triplicates. mRNA libraries were prepared using Novogene NGS RNA Library Prep Set (PT042) and sequenced on the Illumina NovaSeq 6000 system.