title: GSM9654666: mCAF1 H3K27ac input; Mus musculus; ChIP-Seq
library_name: GSM9654666
library_construction_protocol: Briefly, 7-10 × 106 cells at subconfluency were fixed in 1% formaldehyde, washed, lysed and sonicated using the Bioruptor Pico sonication device (Diagenode; B01060001) using 10 cycles (30 sec on; 30 sec off) at maximum intensity. Purified chromatin was then separated for: (i) immunoprecipitation using Dynabeads Protein G (Life Technologies: 10003D) coated with 10 µg of Rabbit anti-Histone H3 acetyl K27 antibody (Abcam, ab4729) per ChIP experiment; (ii) non-immunoprecipitated chromatin, used as Input control; and (iii) assessment of sonication efficiencies using a 1% agarose gel. ChIP DNA was washed, eluted and treated with 2.7 μL of 1 mg/mL RibonucleaseA (RNaseA) for 30 min at 37 °C and subsequently incubated with 1.3 μL of 20 mg/mL proteinase K (Invitrogen) for 1 h at 55 °C. Then DNA extraction was performed using SPRI magnetic beads (Beckman Coulter, B23318). After elution in TE buffer, DNA was quantified using Qubit (ThermoFisher Scientific; Qubit 3.0 Fluorometer; #Q33216) high sensitivity assay (ThermoFisher Scientific; #33216) Prior to sequencing, ChIP samples were library prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, NEBNext Ultra II DNA library prep kit for Illumina, #E7770, NEBNext Multiplex Oligos for Illumina, #E7335L). Adaptor ligated DNA was size selected with SPRI magnetic beads (Beckman Coulter, B23318) for the Illumina sequencer (#NextSeq500). After library preparation, we performed qPCR, high sensitivity DNA quantification and size selection measurement (Agilent Bioanalyzer 2100 system + High sensitivity DNA measurement assay; 5067–4626) before sending samples for sequencing.
identifiers.namespace: GEO
identifiers.id: GSM9654666_r1