high-throughput sequencing data of influents and effluents of five WWTPs in XIAMMEN,FUJIAN

Wastewater collection A total of 30 samples including 15 influents (INF) and 15 effluents (EFF) were collected from 5 WWTPs in Xiamen, China in June 2021 (Table S1). According to turbidity, 1 L of INF was sampled, and 10 L of EFF was collected. To exclude the influence of weather, sampling was performed if there was no rain in recent days. Collected samples were stored in sterilized containers and delivered on ice to the laboratory within 2 h. To analyze the composition of microbial communities, 200 mL INF and 1000 mL of EFF were filtered with 0.22 μm cellulose nitrate membranes (Merck Millipore, USA). The filters were stored at -80 ℃ until the extraction of DNA. These 30 samples were raw water collected from WWTPs without any other treatment and we called them RAW, which contained both free-living bacteria and the intracellular bacteria of the protists.Protists sorting by flow cytometry Protists were sorted from all 30 collected water samples by an S3e Cell Sorter (Bio-Rad, USA) equipped with a 100 μm diameter nozzle. The microbial biomass in effluents was much lower than that in influents, therefore 800 mL of INF and 9 L of EFF were used to obtain enough protists for downstream analysis. In detail, water samples were first filtered through a sterile nylon 100 μm cell strainer (Solarbio, China) to avoid plugging the cell sorter. Then, the filtrate was filtered with 0.8 μm membranes (De Vargas et al., 2015) to collect protists. The collected protists were rinsed into 5 mL Neff’s Modified Amoeba Saline (NMAS) buffer (Geisen et al., 2021) from 0.8 μm filters and then stored at 25 ℃ for 2h. Gentamicin with a final concentration of 300 μg/mL was added into NMAS buffer to kill the remaining extracellular bacteria of protists (Maal-Bared et al., 2019). Subsequently, the protists in NMAS buffer were incubated at 37 ℃ for 10 min with a pH-sensitive green fluorescing probe Lysotracker Green DND-26 (75 nmol/L; Invitrogen, USA) (Rose et al., 2004). Unstained samples were used as a control to set up the cell sorter so that all protists could be easily visualized on forward scatter (FSC) and side scatter (SSC) plots. Before sorting, the cytometer was cleaned thoroughly with bleach, sterile tubes and buffers were used to remove contamination. Both influents and effluents were sorted using the Bio-Rad S3e, targeted cells were identified and sorted using S3e flow cytometer equipped with a 488 nm laser for excitation with peak emission occurring at 511 nm. Protists stained with Lysotracker Green DND-26 were recognized by the green fluorescence (FL1), relative size (FSC), and internal complexity (SSC) using a purity yield mode of Bio-Rad S3e. Data were analyzed using FlowJo software (version 10, Tree Star). Finally, sorted protists were filtered through 0.8 μm membranes and stored at -80 ℃ till DNA extraction. These 30 samples enriched by flow cytometry were called FCM and intracellular bacteria were co-sorted by the cell sorting of their protist hosts. Compared to RAW, intracellular bacteria were greatly enriched in FCM.The difference between v1 and v2 is that we add the doi of the published paper related these data.