title: GSM8701096: Patients-Late_group_ATAC-seq#2; Homo sapiens; ATAC-seq
library_construction_protocol: Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood using a Ficoll–Histopaque gradient (1.077 g/mL; GE Healthcare Life Sciences, Piscataway, NJ, USA). After isolation, cells were resuspended in a freezing medium containing 50% fetal bovine serum (FBS), 10% dimethyl sulfoxide (DMSO), and 40% RPMI-1640 (Invitrogen, Carlsbad, CA, USA) and stored in liquid nitrogen until use. For CD8+ T-cell purification, cryopreserved PBMCs were thawed and labeled with fluorescently conjugated antibodies specific to CD8. Cells were subsequently sorted using the FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA). The purity of sorted populations was verified to exceed 95%. ATAC-seq library was constructed using viable sorted CD8+ T cells (50,000 cells per reaction). Cells were washed with 1× cold Dulbecco's phosphate-buffered saline (DPBS) at 4°C and pelleted by centrifugation at 500 × g for 5 min at 4°C. The pellet was resuspended in 50 μL of cold lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, and 0.01% Digitonin) and incubated on ice for 3 min. Following lysis, 1 mL of wash buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20) was added. Nuclei were isolated by centrifugation at 500 × g for 10 min at 4°C and resuspended in the transposase reaction mix (25 μL 2× TD buffer, 16.5 μL 1× cold DPBS, 2.5 μL Tn5 transposase [Illumina], 0.5 μL 0.1% Tween-20 [Roche], 0.5 μL 0.01% Digitonin [Promega], and 5 μL nuclease-free water). The reaction was incubated at 37°C for 45 min on a thermomixer set at 1,000 rpm. Post-transposition, DNA was purified using a Qiagen PCR cleanup kit. Library amplification was performed with indexing primers from the Nextera kit (Illumina, San Diego, CA, USA). The optimal number of PCR cycles for each sample was determined via qPCR to avoid over-amplification. Final libraries were quantified using a Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and verified with the KAPA Library Quantification Kit (Roche Applied Science, Basel, Switzerland). Indexed libraries were pooled and sequenced on an Illumina NextSeq6000 platform using a paired-end 150 bp read strategy.
identifiers.namespace: GEO
identifiers.id: GSM8701096_r1