Pixul multi sample sonicator

The ChIP–qPCR test was carried out following the instructions provided by Pierce™ Agarose ChIP Kit (ThermoFisher Scientific, CA, USA). Briefly, cells were crosslinked in formaldehyde for 20 min and then stopped using stop buffer. After fixation, quenching, and washing the crosslinked cell pellet, the chromatin was extracted and sheared using the PIXUL™ Multi-Sample Sonicator (Active Motif, Carlsbad, CA, USA). The immunoprecipitation was performed using 3 μg of anti-HIF-1α antibody (ab195352, Abcam, UK) or control IgG (Abcam, Cambridge, MA, USA). After that, ChIP-qualified protein G agarose beads were used to retrieve antibody–antigen complexes. A DNA column elution solution was used to elute the DNA. qPCR analysis was performed on the isolated DNA.

Quadriceps muscles
were removed and snap-frozen in liquid nitrogen and stored at −80
°C. After pulverizing the muscle using an ice cold metal mortar,
10 mg was resuspended in lysis buffer (5% SDS, 10 mM TCEP, and 0.1
M TEAB) and lysed by sonication using a PIXUL multi-sample sonicator
(Active Motif, CA, USA) with pulse set to 50, PRF to 1, process time
to 20 min, and burst rate to 20 Hz. Lysates were incubated for 10
min at 95 °C, alkylated in 20 mM iodoacetamide for 30 min at
25 °C, and proteins digested and purified using S-TrapTM micro
spin columns (Protifi, NY, USA) according to the manufacturer’s
instructions.