title: GSM8947624: memory CD4, PBMCs, H3K27Ac, patient3; Homo sapiens; OTHER
library_name: GSM8947624
library_construction_protocol: Freshly obtained nasal polyps were immediately minced and incubated in RPMI 1640 medium containing 1 mg/ml collagenase, 0.5 mg/ml hyaluronidase, and 0.2 mg/ml DNase I (Sigma-Aldrich). After incubation, nasal polyp mononuclear cells were purified by density gradient centrifugation using Percoll solution (GE Healthcare). PBMCs were isolated from peripheral blood using Ficoll-Paque Plus solution (GE Healthcare). 5×10^4 cells were pelleted, washed with 50 μl 1× PBS, and lysed with 50 μl lysis buffer. The nuclei were re-suspended in a 40 μl transposition reaction with 2 μl Tn5 transposase (Illumina) to tag and fragment the accessible chromatin at 37 °C for 30 min. The CUT&Tag assay was performed using the CUT&Tag Assay Kit (Cell Signaling Technology), according to the manufacturer's protocol. A total of 1×10^5 sorted CD45RO+CD4+ T cells from nasal polyps and PBMCs of ECRS patients Th2 cells were subjected to CUT&Tag using an anti-H3K27Ac antibody (D5E4, Cell Signaling Technology). Fragmented DNAs were purified using the QIAGEN MiniElute PCR Purification Kit and amplified using 8-10 PCR cycles based on the amplification curve. CUT&Tag DNA libraries were prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems kit (Cell Signaling Technology) with 13 cycles of PCR amplification. The libraries were sequenced on an Illumina NextSeq 500 and NovaSeq 6000 Sequencing System.
identifiers.namespace: GEO
identifiers.id: GSM8947624_r1