GSM9317292: CUT&Tag of HCASMC: H3K9ac, DDIT4_OE, replicate 1; Homo sapiens; OTHER
Chromatin extraction and tagmentation were performed using the NovoNGS CUT&Tag 4.0 High-Sensitivity Kit. Briefly, 2×10⁵ cells per sample were immobilized on Concanavalin A-coated magnetic beads, permeabilized with digitonin, and incubated overnight at 4°C with specific antibodies: H3K9ac (Cell Signaling Technology, #9649) or H3K27ac (Abcam, ab177178). Following secondary antibody incubation, tagmentation was carried out using pA-Tn5 transposase. Fragmented DNA was subsequently extracted using Tagment DNA Extract Beads. Libraries were constructed following the manufacturer's protocol for the NovoNGS CUT&Tag 4.0 High-Sensitivity Kit. The extracted DNA was amplified in a 50 µL PCR reaction containing 20 µL DNA, 2.5 µL each of i5 and i7 indexing primers, and 25 µL 2×HiFi Amplimix. PCR products were purified using NovoNGS DNA Clean Beads. Final libraries were quality-controlled and sequenced on an Illumina NovaSeq X Plus-25B platform with 150 bp paired-end reads.