DNA methylation study of satellite cells from the rotator cuff and gastrocnemius muscles of mice

The samples consist of cells from two muscle types in mice – rotator cuff and gastrocnemius muscles. The aim of the project is to study the methylation differences between the two muscle types, specifically in genes involved in adipogenesis and muscle regeneration in the rotator cuff muscle with the gastrocnemius muscle being the control. Specimens from rotator cuff and gastrocnemius muscles were harvested from inducible transgenic Pax7CreERT2:R26RtdTomato mice. After muscle digestion, cells were isolated. Muscle satellite cells expressing tdTomato were collected via fluorescence activated cell sorting. These sorted satellite cells from the rotator cuff and gastrocnemius samples were then used for ERRBS. Enhanced Reduced Representation Bisulphite Sequencing (ERRBS) was performed as described in Akalin et al., 2012., with modifications to account for the lowest amount of gDNA (2.5ng) available for library “RCSC4”. The modifications are as follows: universally methylated non-indexed adapters were used for ligation, and the gel size selection was done in one fragment from 150 to 500pb. Each library was sequenced as a single lane on the HiSeq 2500. ERRBS analysis was carried out on all these samples.