title: Illumina NovaSeq 6000 paired end sequencing; Genome-wide occupancy of H3K27me3 in human gingival mesenchymal and periodontal ligament stem cells in response to Electronic-cigarette
design: Genome-wide occupancy of H3K27me3 in human gingival mesenchymal and periodontal ligament stem cells in response to Electronic-cigarette
library_construction_protocol: GMSCs were separated from the surface of the middle third of the root, and were digested with α-minimum essential medium (α-MEM, Gibco, Carlsbad, CA, USA) containing 3 mg/mL collagenase (type I) and 4 mg/mL dispase (both from Sigma-Aldrich, Billerica, MA, USA) for 15 min at 37 °C. Single-cell suspensions were obtained by passing the digested tissues through a 70-μm cell strainer (BD Bioscience, Franklin Lakes, NJ, USA). Then the cell suspensions were seeded into 10-cm culture dishes (Corning, USA) containing α-MEM supplemented with 15% fetal bovine serum (FBS, Hyclone, New York City, NY, USA), and 1 x GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2, and medium was changed twice a week. Cells were used at passages 3-5 for subsequent experiments. SP600125 (MedChemExpress LLC, Shanghai, China) were dissolved in dimethyl sulfoxide (DMSO). Unless otherwise indicated, cells were pre-incubated with serum-free medium containing same dose of DMSO (0.2%) or 10 μM SP600125 for 2 h Genomic DNA was sonicated into 200 bp around fragments, and incubated with the antibody of H3K27me3 (A2363, Abclonal) at 4 oC overnight. This was followed by incubating for 2 h with Protein A beads (Thermo Fisher Scientific) at 37 °C to pull down the bound DNA fragments. We then isolated the DNA fragments from the pull-down by ethanol precipitation. For high throughput sequencing, we added 3'-dA overhang to the H3K27me3 enriched or input DNA and ligated them to the adapter to build a DNA library.