Hiseq 2500 platform

The hypervariable V4-V5 region of the 16S rRNA gene was amplified using the primer pair (515 F: 5′-GTGCCAGCMGCCGCGG-3′ and 907R: 5′-CCGTCAATTCMTTTRAGTTT-3′ with sample-identifying six-nucleotide barcodes) [17 (link)]. The 4 × 50 μL reaction system was set up for each PCR amplification under the following program: initial denaturation at 95 °C for 5 min, and 30 cycles at 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 10 min. The resulting amplicons were purified, quantified, pooled, and sequenced on an Illumina MiSeq PE300 platform (Novogene, Beijing, China). For metagenome sequencing, approximately 3 μg of sewage DNA was used for shotgun library construction with an insert size of 300 bp, followed by Illumina paired-end sequencing on the HiSeq 2500 platform (Novogene, Beijing, China).

Total RNA (8 μg) of each sample was processed to remove the ribosomal RNA using the Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA). Then, the rRNA-depleted RNA sample was used for the library construction using the NEBNext^® Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, USA), as described (Zhang et al., 2018 (link)). Small RNA sequencing libraries of the two rabbit breeds were constructed using the Illumina TruSeq Small RNA Sample Preparation Kit v2, as described (Chen et al., 2019b (link)). Both stranded transcriptome sequencing and small RNA sequencing were performed on the HiSeq 2500 platform in NovoGene (Beijing, China).

The open reading frame (ORF) sequence of the Drosophila Fzr gene was subcloned into pMT-V5-HisA vector for gene overexpression in Drosophila S2 cells. At 48 h after vector transfection into S2 cells following an induction of 500 μM CuSO₄, total RNA samples were separately prepared from Fzr-overexpressed S2 cells and the control. Three biological replicates were performed. Each of total RNA samples was then sequenced on a HiSeq 2500 platform (Novogene). All raw sequence reads were mapped to the Drosophila genome assembly BDGP6 by using the Hisat2 software. Gene expression levels were evaluated by using the FPKM (fragments per kilobase of transcript sequence per millions base pairs sequenced) values. Differential expression analysis was performed by using the DESeq2 package (44 (link)). The resulting P-values were adjusted by using the Benjamini and Hochberg's approach for controlling the false discovery rate. Differentially expressed genes were determined with the threshold criterion of an adjusted P value of <0.05. All raw data in this study have been uploaded to the Sequence Read Archive of the National Center for Biotechnology Information (NCBI) database (accession number: PRJNA509304).

Fresh leaves of three Lonicera species, Lonicera nervosa Maximowicz, Lonicera ferdinandi Franchet, and Lonicera hispida Pallas ex Schultes, were collected from Chunxin and Huating counties in Gansu province, China, in 2017. The dried plant samples and voucher specimens were deposited in the Key Laboratory of Resource Biology and Biotechnology in Western China (Shaanxi, China). The total genomic DNA was extracted from about 5 g of leaf tissue using a DNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. About 5 ug purified DNA was used to construct paired-end libraries with 350 bp insert size and to sequence on an Illumina HiSeq 2500 platform by Novogene (Beijing, China). We downloaded the other four published Lonicera plastid genome sequences (Lonicera japonica Thunberg in Murray, Lonicera fragrantissima var. lancifolia (Rehder) Q. E. Yang Landrein, Borosova & J. Osborne, Lonicera stephanocarpa Franchet, and Lonicera tragophylla Hemsley) to employ the comparison analysis.

Pseudourostyla cristata cells were separated from a freshwater pond in Taipingjiao Park (120°22′2.12″, 36°3′31.7″) in Qingdao, China. Species was identified through morphological features and the SSU-rRNA gene. The SSU-rRNA gene sequence was deposited into the NCBI Sequence Read Archive with the accession number PP132854. Ten cells were collected, washed with distilled water, and incubated in cell culture flasks with 1% lettuce juice medium and Klebsiella pneumoniae as food resource at 25°C until reaching ∼200 cells/mL^− 1. Cell harvest was performed by centrifugation at 200 g for 3 min. Genomic DNA was extracted using the phenol/chloroform/isoamyl alcohol method. Total RNA was extracted using the TRIzol Reagent (Invitrogen).
One DNA library and one RNA library were constructed with NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, #E7370L, USA) and NEBNext Ultra RNA library prep kit for Illumina (NEB, #E7530S, USA), respectively. Sequencing was performed on an Illumina Hiseq2500 platform with paired-end 150 bp read length at Novogene (Novogene, Beijing, China).

Total RNA was extracted using an RNA extraction kit, following the manufacturer’s protocol (Biotech, Beijing, China). The integrity, quantity, and purity of each RNA sample were examined by 1% agarose gel electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and a NanoDrop^TM 1000 (Thermo Fisher Scientific, Waltham, MA, USA). Ten equimolar high-quality RNA samples for each time point were mixed and sent to Novogene Co., Ltd. (Beijing, China) for cDNA library preparation. Finally, four libraries were sequenced on the Illumina HiSeq 2500 platform for paired-end sequencing with a read length of 125 bp (Novogene, Beijing, China).

The hyper-variable V3–V4 region of the 16S rDNA gene was amplified by PCR using primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′). The PCR products were purified and quantified using a QuantiFluorTM fluorometer (Promega, Madison, WI, USA). Negative controls for PCR amplification were reactions without DNA. The PCR products were purified and quantified using a fluorometer (QuantiFluor; Promega, Madison, WI, USA) and then sequenced on a HiSeq 2500 platform using the PE250 model (Novogene, Beijing, China). The qualified DNA samples were randomly sheared to a length of approximately 350 bp using a Covaris ultrasonic crusher (Woburn, MA, USA). The entire library was prepared using the following steps: end repair, adding a 3′ poly-A tail, ligating adapters, purification, and PCR amplification. Library quality was assessed on a Qubit 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA). The library was sequenced on the Illumina HiSeq platform (San Diego, CA, USA). The sequencing produced an average of 540 million reads (approximately 12 GB) per sample.