GSE173428
Genome-wide identification of Scx transcriptional target genes in tendon development II
Despite their important roles in the musculoskeletal system, tendon and ligaments are much less studied comparing to bone, cartilage and muscle. The lack of knowledge in tendon biology severely hinders the understanding of the etiologies of tendon related diseases and development of efficient clinical treatments. In mouse, Scx gene, encoding a Twist family bHLH transcription factor, is expressed in progenitors of all tendons and ligaments, as well as in mature tenocytes. Previous studies show that inactivation of Scx gene results in absence or severe hypoplastia of force transmitting tendons, with muscle anchoring tendons and ligaments are less affected. Here we report a set of ChIPseq data from E13.5 forelimbs of novel Scx-2xFlag tagged mice in which a Scx-Flag fusion protein is expressed recapitulating endogenous Scx expression, and a set of RNAseq data of Scx-GFP positive cells from E13.5 Scx wildtype, E15.5 Scx wildtype and homozygous mutant forelimbs. Using RNAseq and ChIPseq assays, we identify several genes exhibiting Scx-dependent tendon expression druing differentiation, with Scx binding peaks located within their promoter/enhancer regions. Thus these genes may play critical roles in mediating Scx regulated tendon cell differentiation. Our results provide new insights in the mechanisms of tendon development. Overall design: We generated mice expressing a 2xFLAG epitope-tagged endogenous Scx protein using the CRISPR/Cas9-mediated genome editing technology. 10 pairs of E13.5 forelimb samples were pooled together as a single biological replicate, and 3 replicates were used for ChIP with anti-FLAG antibodies (Sigma #1804). Sequencing libraries were generated using Rubicon ThruPLEX DNA sequencing kit (Rubicon Genomics, Ann Arbor MI). Sequencing was performed on an Illumina NextSeq 500 (Illumina, San Diego CA). Raw FASTQ files were aligned to mm10 reference genome. FACS sorted GFP positive cells from E15.5 Scx wildtype and homozygous mutant embryos were used for total RNA preparation. 3 pairs of controls and mutants were included. Sequencing libraries were generated by using Illumina Nextera DNA Sample Prep Kit and sequenced using Illumina NovaSeq 6000. Sequenced reads were mapped to the reference mouse genome (mm10).