GSM9037208: IgG,NSC197,rep1; Homo sapiens; OTHER
Patient-derived lines were acquired and propagated as previously described. CUT&RUN was performed using a magnetic bead-based protocol using the CUTANA Kit (EpiCypher Cat.14-1048) according to the manufacturer's instructions. The magnet was placed on ice for all steps. Cells were harvested by centrifugation (300g for 3 minutes at room temperature in a swinging bucket rotor) and washed in ice cold Wash Buffer. One million cells were added to low-binding PCR tubes containing activated beads (in triplicate) for 10 minutes at room temperature. Cell Permeabilization Buffer consisted of Wash Buffer containing 0.01% Digitonin. Antibody Buffer consisted of Cell Permeabilization Buffer containing 0.5M EDTA). 0.5ug of each antibody (SOX2-test, H3K4me4-positive control, and IgG-negative control) was added to respective tubes and placed on a nutator overnight at 4ºC for incubation. Extracted DNA fragments were quantified using a QubitTM fluorometer and stored at -20ºC until library preparation was initiated. Library preps were conducted using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina according to the manufacturer's instructions. Sequencing libraries were prepared by amplifying DNA fragments using a 2-step PCR using primers as follows: R1: 5'-[insert]-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC...-3' and R2: 5'-[insert]-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT...-3'. The resulting libraries were subsequently sequenced on an Illumina HiSeq2500 (RRID: SCR_016383).