Universal genomic dna kit

Total DNA was extracted by the Universal Genomic DNA Kit (CWBiotech). Mitochondrial gene Cox1 and nuclear gene Pecam were selected as the representative genes of mitochondrial and nuclear DNA, respectively. Quantitative PCR was used to detect the copy number changes of Cox1.

According to the manufacturer's protocols, under aseptic conditions, genomic DNA was extracted from plasmodia using the Universal Genomic DNA Kit (CWBIO, China) according to the manufacturer's protocols. About 200 mg of plasmodia (collected from several individual plasmodia) were used for DNA extraction. DNA purity was examined on 1% agarose gels. NanoPhotometer spectrophotometer was used to detect sample purity; Qubit2.0 Flurometer was used to measure DNA sample concentration. The V3-V4 hypervariable region of the bacterial 16S ribosomal RNA gene was amplified by PCR (94 °C for 2 min, followed by 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 10 min) using primers 341F 5'–CCTACGGGNGGCWGCAG–3' and 805R 5'–GACTACHVGGGTATCTAATCC–3' [27 (link)], where the barcode was an eight-base sequence unique to each sample. PCR was performed in triplicate with 50 μL mixture containing 5 μL of 10 × Ex Taq Buffer (Mg²⁺ plus), 4 μL of 2.5 mM dNTPs, 1 μL of each primer (20 μM), 0.25 μL of TaKaRa Ex Taq (5 U/μL), and 2.5 ng of the template DNA. Amplicon sequencing targeting 16S V3–V4 region was performed by Annoroad company based on an Illumina HiSeq platform. Roughly 50,000, 250 bp paired-end reads were generated for each sample. Original data have been deposited into the NCBI SRA database with the accession number PRJNA600342.

We sampled 52 populations, comprising a total of 409 individuals between 1999 and 2018 across the natural distributional range of A. paludum in Eurasia. The latitude and longitude of each collection site were recorded using a handheld global positioning system (GPS) unit. All samples were preserved in 70–95% ethanol and stored in a freezer at −20 °C in the College of Life Sciences at Nankai University (NKU, Tianjin, China). Genomic DNA of each specimen was extracted from the entire body, excluding the abdomen and genitalia, of each specimen using a Universal Genomic DNA Kit (CWBIO). Detailed information for molecular voucher specimens is provided in supplementary tables S1–S4, Supplementary Material online, which includes sample codes, sample sizes, collection locations and data, longitude, and latitude.

After 72 h transfection, cells expressing green fluorescent protein were sorted out by flow cytometry (BD FACSAria Fusion). Genomic DNA was extracted using the Universal Genomic DNA Kit (CWBiotech) following the manufacturer’s protocol. The genomic region flanking the target site was amplified by PCR with GoTaq Green Master Mix (Promega). Equal amounts of PCR product were sent for NGS at the Hi-TOM platform.^{71 (link)} Indels were analyzed by the Hi-TOM platform (http://121.40.237.174/Hi-TOM/). The primers used in this study are provided in Supplementary information, Table S5.

Total DNA was isolated from cells using the Universal Genomic DNA Kit (Cwbiotech). Isolated DNA was used for PCR using a Multiplex PCR MasterMix (Cwbiotech). The primers are listed in Supplementary Table 3.
Total RNA was isolated from cells using the RNApure Tissue & Cell Kit (Cwbiotech). Isolated RNA was used as a template for reverse transcription reactions using a HiFiScript cDNA Synthesis Kit (Cwbiotech).
Quantitative real-time PCR analyses were performed with a PCR mixture containing 1 μmol/L of each primer and SYBR Green Real-Time PCR Master Mixes (Thermo Fisher) using a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). Each sample was run in triplicate, and the mRNA level of GAPHD was used as an internal control. The cycle threshold (Ct) value was normalized to the GAPDH level using the 2^−△△Ct method. The primers are listed in Supplementary Table 4.