GSM4681520: LMNA-P17-WT-2; Mus musculus; ChIP-Seq
Frozen hindlimb muscles were crushed to powder and crosslinked in 10mL of PBS with 2% formaldehyde (Sigma, F8775) for 15 minutes at room temperature under rotation. Crosslinking was stopped with 1.5mL of 2.5M glycine. Samples were washed with PBS and incubated on ice for 10 minutes in Farham lysis buffer (5mM PIPES, 85mM KCl, 0.5% N-40, pH 8.0) before bead lysis in Precellys Evolution (3x20s at 6800rpm). Lysates were then incubated on ice for 20min, and the supernatant was removed after centrifugation (1000g, 5 minutes). The remaining pellet was resuspended in TE and 0.2%SDS (10mM Tris-HCl pH 8.0, 1mM EDTA) and nuclei were sonicated on Bioruptor pico (Diagenode) for 10 cycles (30s on, 30s off). 1% of sheared DNA was saved for input and the rest was diluted 1:1 with in 1xTE 0.1% sodium deoxycholate, 1% Triton X-100 and added 8µg of Lamin A/C antibody (Santa Cruz, sc-7292 X) conjugated to 60µl of Protein G Dynabeads (ThermoFisher, 10003D). Lamin A/C immunoprecipitation was performed for 48 hours at 4ºC and beads were subsequently washed twice with RIPA buffer, 360mM NaCl RIPA buffer, LiCl buffer (250mM LiCl, 0.5% NP40, 0.5% deoxycholate, 1mM EDTA, 10mM Tris-HCl, pH 8.0) and TE buffer. DNA was released from the beads by addition of decrosslinking buffer (TE 0.3% SDS, 2mg/mL proteinase K) and incubation at 65ºC for 16 hours under constant mixing. RNA was then removed by incubation with 0.1µg/µl of RNase A and incubation at 37ºC for 1 hour under constant mixing. DNA was purified from the supernatant with Qiagen PCR purification kit (Qiagen, 28104). Standard Illumina library preparation
GEO Accession
GSM4681520