Biophotometer plus spectrophotometer

For the osteoblast gene expression analysis, total RNA was extracted from cultured bone cells by using the NucleoSpin RNA purification kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. The concentration and purity of RNA extracts were determined by using a Biophotometer Plus spectrophotometer (Eppendorf, Hamburg, Germany).

Standard M. tuberculosis strain H37Rv (ATCC 27294) was cultured in Difco Middlebrook 7H9 Broth (BD Biosciences) supplemented with 1:9 volume of oleic acid-albumin-dextrose-catalase and 0.05% Tween 80 (Merck Millipore) at 37°C with 5% CO₂. For experiments, the suspension of H37Rv in the midlog phase was centrifuged at 1,500g at 25°C for 5 minutes and resuspended in serum-free DMEM containing 0.05% Tween 80. After grinding 30–50 times, the achieved homogenate containing a single bacterium was centrifuged again at 1,500g at 25°C for 5 minutes, and the obtained supernatant was detected using Biophotometer Plus spectrophotometer (Eppendorf) at OD 600 nm. The concentration of bacterium suspension with OD value detected at 600 nm of 0.207 was regarded to be 4 × 10⁶ colonies/mL. Varied MOI of H37Rv was used as indicated in different experiments. For detection of intracellular M. tuberculosis load, CFU assays were performed as previously reported (55 (link)). To compare the effects on intracellular M. tuberculosis survival in the face of different phagocytosis amount, the amounts of M. tuberculosis in WT or Znfx1^–/– BMDMs at different times postinfection were normalized to the average M. tuberculosis amount in each type of cells at 0 hours.

Mosquito larvae were immobilized by removing excess water and then injected at the lateral center of the mesothorax. Mosquito adults were cold anesthetized and then injected at the thoracic anepisternal cleft. Finely pulled glass capillary tubes were used as needles, and 0.2 μl of the following substances, alone or in combination, were injected: 0.08 % solids 1-μm diameter green fluorescent (505/515) carboxylate-modified microspheres (Invitrogen, Carlsbad, CA, USA) in phosphate-buffered saline (PBS) (pH 7.0), GFP-expressing E. coli (modified DH5α) in Luria-Bertani’s rich nutrient medium (LB), LB medium, 1 mg/ml pHrodo Red E. coli Bio Particles (560/585 nm; Invitrogen) in PBS, or cellular staining solutions (see the next section). For bacterial infections, E. coli were grown in LB overnight at 37 °C in a shaking incubator until cultures reached approximately OD₆₀₀ = 5, as measured using a BioPhotometer Plus spectrophotometer (Eppendorf AG, Hamburg, Germany).

Total RNA was isolated from leaves using RNAiso Plus (Takara, Dalian, China). Dried RNA samples were dissolved in DEPC-water to 1 μg/μL using a BioPhotometer Plus spectrophotometer (Eppendorf, Hamburg, Germany). RNA was reverse-transcribed using a Takara PrimeScript^® RT reagent kit with a gDNA eraser according to the manufacturer’s specifcations. qRT-PCR was performed using a RealMasterMix (SYBR Green) kit (Tiangen, Beijing, China) according to the manufacturer’s specifications. SYBR Green PCR cycling was performed using an iQ™ 5 multicolour real-time PCR detection system (Bio-Rad, Hercules, CA) with 20 μL samples. PCR primers were designed using Primer Premier 5.0 to avoid conserved regions. Primer sequences are listed in Supplementary Table 1. Wheat TaEF-α (No. Q03033) was used as internal reference gene. The relative quantities were calculated using the 2^−ΔΔCt method^{43 (link),44 (link)}. Each treatment included three replications, and each replication included two technical replications.

Total genomic DNA was extracted using a Genomic DNA Miniprep Kit (Axygen) following the manufacturer’s instructions. The DNA quality was then evaluated by agarose gel electrophoresis and a BioPhotometer Plus spectrophotometer (Eppendorf, Germany).
Subsequently, the two samples were sonicated to produce DNA fragments ranging from 100 to 500 bp. After end repairing, phosphorylating and A-tailing with Paired-End DNA Sample Prep kit (Illumina, USA), the DNA was ligated to an Illumina sequencing primer adaptor. Then the fragments were used for methylated DNA immunoprecipitation (MeDIP) enrichment using a Magnetic Methylated DNA Immunoprecipitation kit (Diagenod, Belgium) following the manufacturer’s recommendations and the qualifying DNA was used for PCR amplification. Then bands between 220 and 320 bp were excised from the gel and purified with a QIAquick Gel Extraction Kit (Qiagen, Germany). The products were quantified with a Quant-iT™ dsDNA HS Assay Kit (Invitrogen, USA) using an Agilent 2100 Analyzer (Agilent Technologies, USA). Following qPCR qualification, DNA libraries were sequenced on the Illumina HiSeq 2000 (Illumina, CA, USA) to generate paired-end 50-bp reads by the Beijing Genomics Institute (BGI, China).

Total RNAs from cultured cells were isolated using TriPure kit according to manufacturer's instructions (Roche, USA). The quantity of RNA was measured using the Eppendorf Biophotometer Plus Spectrophotometer. RNA purity was assessed using the absorbance ratio of 260 to 280 nm, where a value of 1.8–2.0 indicated good quality RNA.

Two mitochondrial genes, cytochrome oxidase c subunit 1 (COI) and 16S ribosomal RNA (16S rRNA), were used. DNA was extracted from a dorsal fin clip of each specimen using Gentra Puregene Tissue Kit (QIAGEN, USA), according to the manufacturer’s instructions. Total DNA concentration and quality was quantified using BioPhotometer Plus spectrophotometer (Eppendorf, Germany). Both mitochondrial cytochrome COI and 16S rRNA genes were amplified using primer pairs (Table 2) to validate the species identity of each specimen. PCR reaction and condition for COI gene was performed PageBreakaccording to Abdul Kadir et al. (2015) , whereas PCR amplification for 16S rRNA gene was conducted according to Arai and Wong (2016) . PCR amplicons were purified using QIAquick® PCR Purification Kit (QIAGEN, USA), labeled with BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems Inc., USA), and sequenced bi-directionally on an ABI PRISM 3730xl Genetic Analyzer. Generated sequence trace files were manually edited and assembled using SeqMan Pro application in DNASTAR version 6.0 (DNASTAR Inc., USA). The contig sequences were compared for percentage similarity with the reference sequences in the GenBank using BLAST search. The sequences for both specimens were deposited to GenBank with accession numbers as listed in Table 3.