C1000 thermal cycler instrument

Antibody or ADC solutions (25 µL, 1 mg/mL in PBS) were combined with 50X SYPRO Orange solution (2.5 µL, diluted from 5000X stock with water, Invitrogen, Carlsbad, CA, USA) in a 96-well RT-PCR plate. Samples were then loaded into a CFX96 Real-Time System equipped with a C1000 Thermal Cycler instrument (BioRad, Hercules, CA, USA) and the temperature was equilibrated to 20 °C for 10 min. Fluorescence intensity was measured at 20 °C and the fluorescence intensity is reported as the average measurement value of five samples ± standard deviation.

Genomic DNA of the sediment microbial community was extracted from each sediment sample using the DNeasy PowerSoil kit (Qiagen, Germany) following the manufacturer's instructions. The concentration and quality of the extracted total DNA were examined using a NanoDrop-2000c UV-Vis spectrophotometer (ThermoFisher Scientific, USA) and 1% agarose gel electrophoresis. The amoA genes of comammox Nitrospira were amplified by nested PCR with the primer set A189y (5′-GGNGACTGGGAYTTYTGG-3′)-C576r (5′-GAAGCCCATRTARTCNGCC-3′) and barcoded primer sets CA209f (5′-GAYTGGAARGAYCGNCA-3′)-cob576r on a C1000 Thermal Cycler instrument (Bio-Rad, USA) (24 (link), 51 (link)). The amplification reaction and thermal program of the nested PCR were performed as described in our previous studies (27 (link), 51 (link)). To increase the specificity of amplification, we employed touchdown PCR to perform the second round of nested PCR. During PCR amplification, negative (i.e., without template DNA) and positive (i.e., recombinant plasmid containing the comammox amoA gene) controls were included. The amplified products were confirmed by 2% agarose gel electrophoresis. The purified amplification products were paired-end sequenced (2 × 250 bp) at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) using an Illumina HiSeq 2500 platform (Illumina, USA).

Genomic DNA was isolated from cultured cells with the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics UAB, and Vilnius, Lithuania). Bisulfite modification of genomic DNAs was performed using the EpiJET Bisulfite Conversion Kit (Thermo Fisher Scientific Baltics UAB, and Vilnius, Lithuania) according to the manufacturer’s protocol. ALDH1A2 is associated with a ~1.5 kb CpG island at its 5′ end, overlapping with the transcription start site [13 (link)]. For methylation-specific PCR analysis, we amplified a 200 bp region within the CpG island upstream of the ALDH1A2 transcription start site using primer pairs specific for either methylated or unmethylated bisulfite-modified sequences (Table S2), as previously reported [13 (link)]. PCR was performed on a C1000 thermal cycler instrument (Bio-Rad, Hercules, CA, USA) using 200 ng of a bisulfite-modified DNA template, 1× EpiTaq PCR buffer, 0.3 mM dNTPs, 2.5 mM MgCl₂, 200 ng of each primer, and 1.25 U EpiTaq DNA polymerase (TaKaRa EpiTaq™ HS for bisulfite-treated DNA; Takara, Nagoya, Japan) in a 50 µL reaction for 40 cycles (94 °C, 30 s; 65 °C, 45 s; and 72 °C, 1 min), followed by a 1.8% agarose gel electrophoresis and visualization using a Gel Doc XR molecular imager system (Bio-Rad, Hercules, CA, USA).