GSM8342378: CUTTag_hESC_OE_OTX2_Rep2; Homo sapiens; OTHER
Human ES cells were cultured in Essential-8 medium (Thermo Fisher, Cat# A1517001) containing Matrigel (Corning, Cat# 354277). Cells were dissociated using Versene Solution (Gibco, Cat# 15040066) Hyperactive Universal CUT&Tag Assay Kit (Vazyme, Cat# TD904) was used to construct the CUT&Tag libraries of OTX2-overexpressed hESCs according to the manufacturer's protocol. hESCs that were not transfected with OTX2-overexpression plasmid were used as the negative control. Briefly, 100,000 EGFP-positive OTX2-overexpressed hESCs or control hESCs were washed and resuspended in 100 μL wash buffer before 10 µL of activated Concanavalin A coated magnetic beads were added to each sample and incubated at room temperature (RT) for 10 mins. After that, cells were incubated with 4 µg of anti-flag antibody (Sigma, Cat# F7425) on a rotating platform for 2 hrs at RT. Then, Anti-rabbit IgG antibody (Abcam, Cat# ab6702) was added and incubated at RT for 60 mins. Next, cells were washed three times with Dig-Wash buffer to remove unbound antibodies. After that, pA/G-Tnp incubation was performed on a rotating platform for 1 hr at RT. Similarly, cells were washed three times with Dig-300 buffer to remove unbound pA/G-Tnp protein and resuspended in tagmentation buffer and incubated at 37ºC for 1 hr. Tagmentation was stopped by adding of 10% SDS and incubating at 55 ºC for 10 mins. Then cells were plated on a magnet rack, supernatant was transferred into a new PCR tube and DNA was extracted by using DNA Extract Beads Pro. Finally, libraries were constructed using TruePrep Index Kit V2 for Illumina (Vazyme, Cat# TD202). All CUT&Tag libraries were sequenced using the Illumina NovaSeq 6000 platform with PE150 sequencing strategy.