Rnaprep pure cell bacteria kit

ADSCs that were treated by H₂O₂ with or without Exendin-4 were collected. Total RNA was extracted from cells using RNAprep pure cell/Bacteria Kit (TIANGEN) and the instructions provided by the manufacturer. First-strand cDNA was synthesized using Thermo First cDNA Synthesis Kit (Germany) according to the standard procedures. The qPCRs were performed in triplicate with the FastStart Universal SYBR Green Master (ROX; Roche, Mannheim, Germany) and run on the StepOnePLUS system (Applied Biosystems, USA). Primers of β-actin used as an internal standard were: forward (5′-GCTACAGCTTCACCACCACA-3′), reverse (5′-GCCATCTCTTGCTCGAAGTC-3′). Two main genes that mediated cell adhesion, integrin αV and the integrin β1,were analyzed. The primers of integrin αV were: forward (5′-AATGTCAGCCCAGTCGTGTCTTAC-3′), reverse (5′- CCAACGTCTTCTTCAGTCTC-3′). The primers of integrin β1 were: forward (5′- ACTAAAGTGGAAAGCAGGGAGAA-3′), reverse (5′- GAAATAGAACCAGCAGTCATCAAT-3′). Samples were run in duplicate with RNA preparations from three independent experiments. The qRT-PCR was performed with an Applied Biosystems StepOne PLUS.

DSM 7029 mutant cells were harvested at an OD₆₀₀ value of approximately 4.3 ± 0.5, at approximately 18 h. RNA extraction was carried out with an RNAprep Pure Cell/Bacteria Kit (TIANGEN, China) according to the manufacturer’s instructions. Genomic DNA was removed by using DNA Eraser supplied in the 1st Strand cDNA Synthesis Kit (Takara), and its removal was confirmed by PCR. cDNA was synthesized using HiScript^R III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme Biotech, China) for RT-PCR with 800 ng of total RNA. Gene expression was analysed using real-time RT-PCR with ChamQ™ Universal SYBR^R qPCR Master Mix (Vazyme Biotech, China). PCR was conducted in a BIO-RAD Real-Time System with the following thermal cycling program: 30 s at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. The mraW gene was used as the normalization signal. The amplification primers used for each gene are listed in Additional file 1: Table S3. The transcription levels of fdx_0135, fdr_0130, and fdr_7100 were measured.

EHLJ7 was synthesized by our institute. The structure of EHLJ7 is showed in Figure 1A. Roswell Park Memorial Institute (RPMI) 1640 Medium and Hyclone™ fetal bovine serum (FBS) were both purchased from GE Healthcare (Waltham, USA). Interleukin-6 (IL-6) was obtained from Proteintech Group, Inc. (Chicago, USA). Sodium butyrate (SB) was purchased from Sigma Aldrich (St. Shanghai, China). Anti-JAK2, anti-p-JAK2, anti-STAT3, anti-p-STAT3, and anti-SOCS1 antibodies were purchased from Abcam, Inc. (Shanghai, China). DSS was obtained from MP Biomedicals (USA). Radio-immunoprecipitation assay (RIPA) lysis buffer was purchased from Sorlarbio Bioscience & Technology CO. INC (Beijing, China). Bicinchoninic Acid (BCA) Protein Assay Kit was purchased from Applygen Technologies Inc (Beijing, China). The ELISA kits for tumor necrosis factor alpha (TNFα) and IL-6 were from R&D Systems, Inc. (Minneapolis, USA). RNAprep pure Cell/Bacteria Kit, FastKing RT Kit, and SuperReal PreMix Plus were purchased from Tiangen Biotech (Beijing, China).

To determine the mRNA level of wsv191 and wsv407, quantitative real-time PCR was performed with the wsv191-specific primers (5′-TTGACGAGGAGGATTGTAA AGG-3′ and 5′-ATACCAGGGTTTATTTTGTTGCG-3′) or wsv407-specific primers (5′-AACCCATTCCACCCCAATATC-3′ and 5′-ATATCTTTGTCGGCCAACTTGTC-3′) as described before (19 (link)). Shrimp β-actin was used as an internal control (primers 5′-CGAGCACGGCATCGTTACTA-3′ and 5′-TTGTAGAAAGTGTGATGCCAGAT CT-3′). Total RNAs were extracted from shrimp hemocytes using RNAprep Pure Cell/Bacteria kit (Tiangen Biotech, China). The cDNA was synthesized with PrimeScript^TM 1st strand cDNA synthesis kit (Takara, Japan). Quantitative real-time PCR reaction mixture contained 5 μl of SYBR® Premix Ex Taq, 0.5 μl of 10 μM forward and reverse primers and 100 ng of cDNA template. The PCR reaction conditions were: 95°C for 1 min, followed by 45 cycles of 30 s at 95°C, 30 s at 52°C, and 30 s at 72°C. For each treatment, quantitative real-time PCR was independently carried out for three times to quantify mRNAs.

Northern blotting was carried out to examine the expression levels of miRNA and mRNA (19 (link)). For the miRNA Northern blotting, miRNAs were extracted from shrimp hemocytes using the mirVana miRNA isolation kit (Ambion, USA) according to the manufacturer's instructions. On the other hand, for Northern blot analysis of mRNA, total RNAs were extracted with RNAprep Pure Cell/Bacteria Kit (Tiangen Biotech, China). After electrophoresis for 2 h, the RNAs were transferred onto a nylon membrane (Amersham Biosciences, UK), followed by UV cross-linking. The membrane was pre-hybridized in DIG (digoxigenin) Easy Hyb granule buffer (Roche, Switzerland) for 0.5 h at 42°C and then hybridized with DIG-labeled miR-1000 probe (5′-TACTGCTGTGACGGGACAATAT-3′), U6 probe (5′-GGGCCATGCTAA TCTTCTCTGTATCGTT-3′), wsv191 probe (5′-TTCTTGGCTGCAGTTGAAACCC AGCGAACCCT-3′), wsv407 probe (5′-CTCTCCACCCTTTCAATGATGGTAATGG AAGAAC-3′) or β-actin probe (5′-ATGTCACGAACGATTTCTCGCTCGGCGGTG-3′) at 42°C overnight. In shrimp, β-actin is used as an internal control (19 (link)). The detection was done with the DIG High Prime DNA labeling and detection starter kit II (Roche).

Total RNA was extracted from porcine SCs using the RNAprep Pure Cell/Bacteria Kit (DP430, TIANGEN) according to the manufacturer’s instructions. The quality and concentration of the RNA were detected with a spectrophotometer (NANODROP 2000; Thermo Scientific). Next, cDNA was synthesized by reverse transcription using the PrimeScript^TM RT Reagent Kit with gDNA Eraser (RR047A, Takara) according to the manufacturer’s instructions. RT-qPCR was performed on an Eppendorf AG-5341 instrument (Eppendorf, Germany) using SYBR Green Master Mix (04913914001, Roche). The primers used for RT-qPCR were designed using Primer Premier 5.0 software. The qRT-PCR primers of each gene are shown in Table 1. The relative mRNA expression levels were determined using the 2^−△△Ct method and normalized to TBP.

RNA sequencing was performed as previously described with slight modification^{59 ,60} . K. pneumoniae strains 16–312 and 16–312Δcps mutant were grown in LB to an OD₆₀₀ of 0.7. Bacteria were pelleted by centrifugation, snap frozen in liquid nitrogen and cryopreserved at −80 °C. Total RNA from bacteria was extracted and purified using the RNAprep Pure Cell / Bacteria Kit (TIANGEN Biotech, catalogue number DP430). RNA integrity was assessed using the Agilent 2100 Bioanalyzer. RNA sequencing was performed at the Shanghai Personal Biotechnology (Shanghai, China). P < 0.05 was defined as significant difference. The LPS and peptidoglycan synthetic genes and their putative gene products are listed in Supplementary Table 14.