Total rna purification kit

Extracting RNA from exponentially growing cells is essential for profiling the dynamic alterations in biofilm-related genes during the initial phases of S. aureus biofilm formation (8 (link)). Thus, S. aureus isolates were cultured in TSB at 37°C for 9 h under two conditions: with or without 4 µg/mL YH7. RNA extraction was performed using a Total RNA Purification Kit (Sangon Biotech). The PrimeScript RT Reagent Kit with gDNA Eraser (Takara) was used for reverse transcription. Quantitative PCR analysis was conducted using the QuantStudio 5 Real-Time PCR System with TB Green Premix (Takara). The RNA expression changes of the target genes were calculated using the formula 2−ΔΔ^Ct (31 (link)). The primer pairs used are listed in Table 1. Each reaction was performed in triplicate.

RNA isolation and high-throughput RNA sequencing (RNA-Seq) were conducted by Oebiotech Corp. (Shanghai, China). Total RNA was extracted using a Total RNA Purification Kit (Sangon Biotech, Shanghai, China), followed by evaluating the RNA integrity on Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The libraries were sequenced on an Illumina sequencing platform (HiSeq 2500), and 150-bp/125-bp paired-end reads were generated. The gene expression profiles were analyzed based on reads per kilobase of transcript per million mapped read (RPKM) normalization. DESeq was used to standardize the counts of genes in each sample, and NB was used to test the difference significance of reads. Differential genes were screened according to the difference multiple and different significance test results. Finally, the GO and KEGG enrichment of differentially expressed genes were analyzed under the cutoff of p-value < 0.05 using the BLAST program.

Total RNA was extracted from cells at day 1, 3, 5 and 7 using a total RNA Purification Kit (Sangon Biotech, Shanghai, China) following the supplier’s instructions. Then samples were treated with an RNA Cleanup and Concentration Kit (Sangon Biotech) to ensure RNA samples were free from contaminating DNA. RNA was subjected to reverse transcription using a Moloney murine leukemia virus (M-MuLV) first chain cDNA synthesis Kit (Sangon Biotech). Quantitative real-time PCR was carried out using a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with Power SYBR Green PCR Master Mix (Applied Biosystems). PCR cycling conditions were: pre-denaturation at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at the appropriate temperature (54–60 °C; Table 1) for 30 s and elongation at 72 °C for 30 s. Genes of CD31, VEGF for HUVECs and SM-MHC and α-SMA for HAVSMCs were selected and the human housekeeping gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference transcript. Primer sequences are shown in Table 1. The 2^−△△CT method was used for quantitative analysis. Each sample was analyzed in triplicate.

Total RNAs were isolated from 3 kinds of tissues (leaves, phloem, and xylem from the current-year branches) using a Sangon Total RNA Purification Kit according the manufacturer’s protocol (Shanghai Sangon, Shanghai, China). The libraries were constructed and sequenced with the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA), as described in our previous report [25 (link)]. An index of the reference genome was built using Bowtie v2.0.6, and single-end clean reads were aligned with the reference genome using TopHat v2.0.9 [49 (link)]. Read counts were calculated using HTSeq (https://www.huber.embl.de/users/anders/HTSeq, accessed on 3 June 2019) and normalized to the expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) [49 (link)] in order to obtain the relative expression levels.
In order to annotate the assembled unigenes, homology searches were conducted against seven databases, such as NR (NCBI nonredundant protein database), the KEGG, COG (Clusters of Orthologous Groups of proteins), UniProtKB/Swiss-Prot (UniProt Knowledgebase), the KOG database (EuKaryotic Orthologous Groups of proteins database), Pfam (Pfam protein families database), the Ortholog database, and the GO (Gene Ontology) database.

Gene expression levels of CD31 and VEGF for VECs from regenerative endothelial tissue covered on the inner surface of implanted SFTFs were analyzed by a quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). According to the supplier’s instructions, total RNA was extracted from the scaffolds using a total RNA purification kit (Sangon Biotech, Shanghai, China), then reverse-transcribed using a cDNA synthesis kit (M-MuLV, Sangon Biotech). RT-PCR were performed as follows: 95 °C/15 min for pre-denaturation, 40 cycles of 95 °C/30 s for denaturation, (54–60 °C)/30 s for annealing and 72 °C/30 s for elongation, and carried out on a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers and the annealing temperature of each gene are shown in Table 1. The housekeeping gene GAPDH was used as the reference transcript. The 2^−ΔΔCT method was used for the quantitative analysis.

Biotinylated lncRNA OXCT1-AS1, antisense lncRNA OXCT1-AS1, and lncRNA OXCT1-AS1 fragments were transcribed in A549 and 293T cells. The RNA products treated with RNase-free DNase I (Thermo Fisher Scientific) were purified using a Total RNA Purification Kit (Sangon Biotech Co., Ltd.). Then, 4 μg of biotin-labelled RNAs were denatured (65°C, 300 s) in PA buffer and slowly cooled. Next, the folded RNA was supplemented with 2 U/mL RNasin (Solarbio) and streptavidin Dynabeads (Thermo Fisher Scientific) and then incubated for 60 min at 4°C. After washing with wash buffer (4 × 5 min) and preclearing using streptavidin Dynabeads, the protein lysate was incubated with the folded RNA-bead complex and 20 μg/mL yeast tRNA at 4°C for 210 min. After washing, beads were boiled in 40 μL 1x SDS loading buffer for 600 s. Then, lncRNA OXCT1-AS1 interacting proteins were subjected to western blot analysis.