Dsdna hs assay kit for qubit

DHT (purity ≥ 98.0%), Tan I (purity ≥ 98.0%), CT (purity ≥ 98.0%), Tan IIA (purity ≥ 98.0%), and D-luciferin potassium salt (purity ≥ 98.0%) were purchased from Yuanye Biotechnology (Shanghai, China). PMA, DDP, PTX, Dulbecco′s modified Eagle′s Medium (DMEM), RPMI-1640, 0.25% trypsin-EDTA, fetal bovine serum (FBS), and penicillin/streptomycin solution were purchased from Gibco (Grand Island 14072, NY, USA). Antibodies against β-actin, and goat anti-rabbit were purchased from Proteintech (Wuhan, China). Histone H3 (citrulline R2 + R8 + R17), Mpo, and Ly6G were purchased from Abcam (Cambridge, UK). Matrigel was purchased from Thermo Fisher Scientific (Waltham 02454, MA, USA). The dsDNA HS Assay Kit for Qubit was purchased from Yeasen (Shanghai, China). Crystal violet and ROS assay kits were purchased from Solarbio (Beijing, China).

The forward primer 5′CCTACGGGNGGCWGCAG-3′ and reverse primer 5′GACTACHVGGGTATCTAATCC-3′ targeting the hypervariable V3–V4 region (341F/805R) of the 16S rRNA gene was used for PCR amplification of the extracted DNA. The reaction was performed on a T100Thermal Cycler PCR system (Bio-Rad Laboratories, Hercules, CA, USA) with the following program: 3 min of denaturation at 95°C, followed by 21 cycles of 0.5 min at 94°C (denaturation), 0.5 min at 58°C (annealing), and 0.5 min at 72°C (elongation), with final extension at 72°C for 5 min. PCR products were detected by 1.5% agarose gel electrophoresis. Amplicons were purified using Hieff NGS DNA Selection Beads (YeasenBiotech, Shanghai, China) and quantified using the dsDNA HS Assay Kit for Qubit (Yeasen Biotech, Shanghai, China). According to the measured values, the product was mixed at equal ratios (50 ng) and sent to Shanghai Mobio Biomedical Technology Co. (Shanghai, China) for high-throughput sequencing on the Miseq platform (Illumina, San Diego, CA, USA) according to the standard manufacturer's protocols. The raw read data of all the samples were deposited in the National Center for Biotechnology Information BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/, PRJNA837028).