GSM5127876: 4C_Quadriceps_Myh4; Mus musculus; OTHER
Muscle nuclei were isolated with hypotonic buffer (25mM Hepes-KOH pH 7.8, 10mM KCL, 1.5mM MgCl2, 0.1% NP40, PIC 1X (complete protease inhibitor Roche), PMSF 1mM). Muscles were sliced with a scissor and then homogenized with an Ultra-Turrax. The solution was transferred in a 15 ml Falcon tube and crosslinked with 2% formaldehyde at room temperature during 10 min. 1,43 ml of cold glycine (1M) was added to quench the formaldehyde for 5 min at +4°C while shaking. The crosslinked nuclei were dounced 10 times with a loose pestle and then centrifuged at 1000 Rcf for 10 min at +4°C. The nuclear pellet was resuspended in 5ml of hypotonic buffer and filtered with 70µm and 40µm cells strainers. The nuclei were pelleted with centrifugation at 1000 rcf for 10 min, snap frozen into liquid nitrogen and stored at -80°C. PCR amplification using primers with included Tru-seq adapter sequences and indexes.