Sanger sequencing

The full-length coding sequence of STC2 was cloned into pLV-IRES-eGFP vector, and the shRNA of STC2 was inserted into pLKO.1 vector. The shRNA sequence of STC2 was 5′- AGG GCA AGT CAT TCA TCA AAG C -3′. The constructs were confirmed by Sanger sequencing in Invitrogen. And the packaging and infection of overexpression and knock-down vectors were briefly described as follows: the overexpressing pLV-IRES-eGFP-STC2 or knocking down plasmids pLKO.1-STC2 were, respectively, cotransfected with pCMV-VSV-G and pCMV-deltaR8.91 plasmids into HEK293T cells with lipofectamine 2000 and cultured for 48 h. The supernatant was collected and filtrated through 0.45 μm filter membrane to remove the cells and fragments. Then the collected solution was subjected to lentivirus concentration solution kit according to the manufacturer's instruction. The enriched lentivirus solution was stored in ultralow temperature freezer.

Adult males were sampled daily from day 1 to 30 after emergence (DAE). Total RNA was isolated with TRIzol reagent (Invitrogen) following the manufacturer’s instructions. Total RNA was incubated with 10 U DNase I (Thermo Scientific, USA) at 37 °C for 30 min for mRNA purification. First strand cDNA was produced from 5 μg RNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Target gene sequences (lola, topi, per, aly, rac, rho, upd, magu) were obtained by retrieving previously constructed B. dorsalis transcriptome data (Y.-C. D., Z.-J. W., C.-Y. N., unpublished data), and their gene specific primers were designed using Primer Premier 5.0 (Premier, Canada) (Table S1). PCR amplicons were purified using AxyPrep DNA Gel Extraction Kit (AxyPrep, USA). The purified products were ligated to cloning vector by using pMD™ 18-T Vector Cloning Kit (TaKaRa, China). The plasmid recombinants (pMD-18T-lola, -topi, -per, -aly, -rac, -rho, -upd, -magu) were amplified by PCR and verified by Sanger sequencing (Invitrogen, Shanghai, China).

We created lentiviral constructs to express SOCS3 with C-terminal Myc and DDK tags by subcloning the human SOCS3 coding sequence and tGFP in a single mRNA using a P2A linker following Origene’s TrueORF cloning instructions. Briefly, SOCS3 cDNA was cut with EcoRI and XhoI and subcloned into pLenti-C-Myc-DDK-P2A-tGFP lentiviral gene expression vector. A lysine 6 mutation (K6Q-SOCS3) was obtained by PCR mutagenesis using PfuUltra II Fusion High-fidelity DNA Polymerase. All constructs were confirmed by Sanger sequencing (Genewiz).
Lentiviral particles were grown in HEK293FT cells (Invitrogen, Thermo Fisher Scientific, R700-07) by cotransfecting cells with the SOCS3-expressing lentiviral constructs or pLenti-C-Myc-DDK-P2A-tGFP (empty vector control) and pCMV-dR8.2 dvpr and pCMV-VSVG packaging plasmids (98 (link)). After 48 hours, medium was concentrated using a 30 kDa cutoff filter, aliquoted, and stored at –80°C until use. Viral titer was determined by the proportion of infected cells with green fluorescence 72 hours postinfection.

Blood samples obtained from the proband were sent to the Nanfang Hospital Precision Medicine Center for Whole-exome sequencing (WES) of genomic DNA. Illumina HiSeq platform was used for WES. The criteria established and revised by the American College of Medical Genetics and Genomics (Richards et al., 2015 (link)) was used to classified the variants. Two hypertriglyceridemia associated genes mutations, within exon 10 of the LMF1 gene and exon 5 of the LPL gene identified by WES, were verified using Sanger sequencing. Standard phenol/chloroform extraction was performed to extract genomic DNA from the peripheral blood acquired from the proband and his family members (Ⅰ1, Ⅰ2, Ⅱ4, Ⅱ2, Ⅲ4, and Ⅲ3). The PCR primers that were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast) were as follows: LMF1: Exon-10 forward primer: 5′-CCG​TCT​CAG​CCA​CCA​GAA​AA-3′, Exon-10 reverse primer: 5′-CAC​GGC​TGG​TTT​GGT​TTG​AG-3'; and LPL: Exon-5 forward primer: 5′-CCA​GCC​ATC​CTG​AGT​GGA​AA-3′, Exon-5 reverse primer: 5′-GGCTCTAAGGTGGTCATGCT-3'.The PCR products were then analyzed by agarose gel electrophoresis and submitted to Invitrogen (Shanghai, China) for Sanger sequencing.

To evaluate the quality of the mRNA‐seq data and expression level, six annotated unigenes were selected randomly and amplified using reverse transcripts (RT)‐PCR and were quantified by real time quantitative PCR (qRT‐PCR). Total RNA was extracted from the previous samples and conducted as described above. The primer sets for each unigene were designed using Primer Premier 5.0 software (Tables S1, S2). The cDNA of unigenes was synthesized according to the manufacturer's protocol (PrimeScriptRT reagent Kit; TaKaRa, Kusatsu, Shiga, Japan). The amplified products of RT‐PCR were cloned into pGEM‐T Easy Vector Systems (Promega, Madison, WI) after purified from the gel, then for Sanger sequencing (Invitrogen Biotechnology, Guangzhou, China). All the unigenes sequenced target fragments were aligned to the nonredundant transcripts in mRNA‐seq using BLASTn (E‐value ≤10⁻⁵).
qRT‐PCR was performed using LightCycler^® 480 SYBR‐Green I Master (Roche Diagnostics, Basel, Switzerland) and run on the LightCycler^® 480 Real‐time PCR system (Roche Diagnostics Ltd). Data for each sample were normalized in relation to the internal control gene (β‐actin) using the 2^−ΔΔCT method (Livak and Schmittgen 2001). All samples were tested in triplicate, and the experiments were performed on three biological replicates.

The sequences of four MCMV full-length cDNAs were determined by Sanger sequencing (Invitrogen, Shanghai, China), and the full-length sequences were assembled in SeqMan software (DNASTAR, Madison, WI, USA). The resulting sequences were aligned against all the available full-length MCMV sequences in the GenBank database using the software Mega535 (link). A phylogenic tree was then constructed with the neighbor-joining method provided in Mega5.