GSM5495161: Osteoarthritic chondrocytes1_IgG; Homo sapiens; OTHER
In situ chromatin profiling using CUT & RUN was performed according to Skene et al 34. Briefly samples were FACS sorted using DAPI to select live cells and 10,000 cells were collected in 10%FBS-PBS media. Cell nuclei were immobilized on Concanavalin A beads after washing. pSTAT3 or normal rabbit IgG antibodies (Cell signaling technology) were incubated with the nuclei overnight in the presence of 0.02% digitonin at 4°. The next day, 700ng/mL of proteinA-micrococcal nuclease (pA-Mnase purified in house with vector from Addgene 86973 76) were incubated with the nuclei at 4 degrees for an hour. After washing, the tubes were placed in heat blocks on ice set to 0 degrees, CaCl2 (1mM) was added and incubated for 30 min before 2X Stop buffer containing EDTA was added. DNA was extracted using Qiagen DNA isolation kit according to manufacturer's protocol Purified DNA was quantified in Qubit and Bioanalyzer (2100) traces using D5000 high sensitivity chip were run to determine the size of the cleaved products. UMI-coded libraries were generated using Swift Biosciences-ACCEL-NGS® 2S PLUS DNA LIBRARY KITS according to manufacturer's protocol. Pair-end (75bp) Illumina sequencing was performed on the UMI-coded and amplified libraries using NextSeq platform. Swift Biosciences-ACCEL-NGS® 2S PLUS DNA LIBRARY KITS