GSM9138346: ARPE-19, G1/S, macroH2A1, Rep2 (Chip); Homo sapiens; ChIP-Seq
In brief, collected cells are lysis in hypotonic buffer. After centrifugation, then resuspend in PA buffer. Cells are homogenized with a Dounce homogenizer for 10min with intermittent pauses. After removing large debris, supernatant is filtered through 10 μm and 5 μm filters. Chromosomes are centrifuged at 1750 × g for 10 min at 4°C and resuspend in MNase buffer. Sequencing libraries are generated using approximately 1 ng of DNA per sample with the NEBNext® Ultra™ II DNA Library Prep Kit (E7645S, NEB, Ipswich, MA, USA), and sequencing is conducted on the Illumina® NovaSeq 500 platform. Prior to library preparation, the DNA is extracted using phenol-chloroform and ethanol precipitation. To obtain the DNA, either input mono-nucleosomes or ChIP-purified macroH2A1 nucleosomes are treated overnight at 56°C with 1% SDS and 200 μg/mL Proteinase K (Invitrogen, Carlsbad, CA, USA) following a pre-incubation with 100 μg/mL RNase A (Invitrogen, Carlsbad, CA, USA) to eliminate RNA contamination.