title: GSM8877100: OM2_H3K27ac; Mus musculus; OTHER
library_construction_protocol: CUT&RUN was performed to profile enhancer histone modifications H3K4me1 and H3K27ac from young, old and geriatric MuSCs following guidelines in the CUTANA CUT&RUN Protocol (EpiCypher). Briefly, MuSCs were pelleted, and resuspended in wash buffer. ~10,000 cells per sample were bound to activated Concanavalin A beads (Bangs Laboratories). The bead-bound cells were incubated with antibody in wash buffer supplemented with 2 mM EDTA and 0.01% digitonin overnight at 4ºC, digitonin was added to permeabilize cells for antibody binding in situ. After washing with 0.01% digitonin in wash buffer, CUTANA® pAG-MNase (1:20) was added to each sample and incubated at RT for 10 mins to allow binding to antibody-labeled chromatin. Samples were then equilibrated on ice water for 5 mins. 2 mM CaCl2 was added to each sample to activate MNase and cleave antibody-bound chromatin. After 2 h of incubation with CaCl2 at 4ºC, reactions were stopped by adding 33 µL stop buffer to degrade RNA and release DNA fragments. DNA was purified using the Monarch DNA Cleanup Kit (NEB) following manufacturer's instructions and the concentration of CUT&RUN-enriched DNA was determined using the Qubit fluorometer with a DNA high sensitivity kit. ~2 ng of purified DNA was used to prepare libraries using NEBNext Ultra II library preparation kit with unique dual index PCR primers (NEB). The library concentrations were determined using Qubit fluorometer with a DNA high sensitivity kit. Library quality and quantity were confirmed on a BioAnalyzer (Agilent) high sensitivity DNA chip. Equimolar amounts of each library were combined, and the pooled library was further quantified using a NEBNext Library Quant Kit (NEB). The CUT&RUN libraries were subjected to paired-end sequencing with on the NextSeq 2000 platform using a P2 100 cycle kit (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM8877100_r1