GSM9337308: H3K27ac - NPC neuron treated with LV-Scrb rep1; Mus musculus; OTHER
Cells were gently washed with PBS and scraped into 1 ml PBS. An aliquot equivalent of 600,000 cells were used for each CUT&Tag sample. Cells were collected by centrifugation for 4 min at 300xg and supernatant was discarded. Cells were resuspended in lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 0.5 mM spermidine, 1.5 mM MgCl, 0.1 mM EDTA, and 0.25% IGEPAL CA-630) and incubated for 5 min on ice. Lysis was assisted mechanically by gently pipetting up and down 10 times with 1000 μL tips.  Successful lysis was confirmed by Typan blue staining and inspection under a light microscope. Nuclei were collected by centrifugation for 4 min at 800xg. Nuclei pellet was washed with CUT&Tag wash buffer and resuspended in 1.5 mL wash buffer. All the procedures were performed according to manufacturer's guides. Concanavalin A beads were conditioned by washing with 1.6 mL 1X CUT&Tag binding buffer two times. Prepared beads were resuspended in binding buffer and slowly added to the nuclei suspension. For each sample, 20 μL of beads were used. Nuclei binding was performed for 10 min at room temperature in a rotator. Beads-bound nuclei were collected using a magnetic stand and clear supernatant was discarded. Beads were resuspended in 50 μL ice-cold CUT&Tag antibody buffer and desired amount of antibody was added (2 μL CTCF antibody). Suspension was gently mixed by vortexing. Primary antibody binding was performed overnight at 4°C. After a quick spin, beads were cleared on a magnetic stand and supernatant was discarded. Beads were resuspended in 100 μL of 1:100 diluted secondary antibody in CUT&Tag Dig-Wash buffer and incubated for 1h at room temp with rotation. After secondary antibody binding, three washes with 1 mL CUT&Tag Dig-Wash buffer were performed. Beads were resuspended in 1:100 diluted CUT&Tag transposomes in CUT&Tag Dig-300 buffer and incubated on an orbital rotator for 60 min at room temperature. After transposome binding, beads were washed three times with Dig-300 buffer. Tagmentation was induced by incubating beads in 125 μL of CUT&Tag tagmentation buffer for 1h at 37°C. After tagmentation, total DNA was extracted using columns. Purified DNA was PCR amplified using proper index primers. Then, the PCR reaction was purified using SPRI beads. Samples were pooled equimolar. Sequencing was performed PE150.