Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

Abstract

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

Myod1 and GR coordinate myofiber-specific transcriptional enhancers

“…Nuclei were isolated from mouse skeletal muscle as described ( 34 ). In brief, muscles were homogenized in hypotonic lysis buffer [10 mM HEPES–KOH pH 7.3, 10 mM KCl, 5 mM MgCl 2 , 0.1% NP-40, 0.1 M PMSF, protease inhibitor cocktail (45 μg/ml; Roche)].…”

“…Chromatin immunoprecipitation (ChIP) followed by qPCR analysis (ChIP-qPCR) was assessed on skeletal muscle or C2C12 myotube nuclear extracts as described ( 34 ), using anti-GR (C-terminal, IGBMC, #3249), anti-Nrf1 (Abcam, ab55744), anti-Myod1 (Cell signaling, 13812), or anti-Myog (Santa Cruz, SC576) antibodies, or a mouse or a rabbit IgG negative control on protein G-Sepharose 4B (GE Healthcare) ( 35 ). Primers used for ChIP-qPCR are described in Supplementary Table S3 .…”

“…For ChIP-seq analysis, libraries were prepared from AR- (C-terminal, IGBMC, #3299), GR- (C-terminal, IGBMC, #3249), H3K27ac- (Active Motif, 39133), H3K27me3- (Active motif, 39155), H3K4me3- (Abcam, 1012-100), H3K4me1- (Active Motif, 39297), Polr2- (Santa Cruz H-224, SC9001) and Ctcf- (Sigma-Aldrich, 07–729) immunoprecipitated DNA from skeletal muscle nuclear extracts as described ( 34 ). ChIP-seq libraries were sequenced with an Illumina Hiseq 4000 as paired-end 100 bp reads for Ctcf and H3K4me3 or single-end 50 bp reads for the other sequencing datasets, and mapped to the mm10 reference genome using Bowtie 2 ( 25 ).…”

A fastMyhsuper enhancer dictates adult muscle fiber phenotype through competitive interactions with the fastMyhgenes

“…Nuclei purification from adult skeletal muscle has been performed as previously described 74 with some modifications. After dissection, 16 soleus or 8 quadriceps were resuspended in 1mL of hypotonic buffer (25mM Hepes-KOH pH 7.8, 10mM KCL, 1.5mM MgCl2, 0.1% NP40, PIC 1X (complete protease inhibitor Roche), PMSF 1mM) in a 2ml tube for 5 min at +4°C.…”