Steponeplus real time pcr system

According to the manufacturer's instructions, total RNA was extracted and purified from the left upper lung tissue of each mouse and amplified by polymerase chain reaction (PCR). The constructed library was examined on an Agilent 2100 Bioanalyzer and ABI StepOneplus Real-Time PCR System and sequenced on the Illumina HiSeq platform. The clean reads were obtained by removing low-quality reads, adapters, and poly-N sequences from the raw reads. The clean reads were matched to the reference genome sequence (GRCm38) using HISAT2-software, new transcripts were predicted, and single-nucleotide polymorphism (SNP), insertion-deletion (InDel), and differential splicing genes (DSG) were identified. The new transcript with protein coding potential was added to the reference gene sequence to form a complete reference sequence and then the gene expression was calculated. Finally, the quality of data from each sample and the differentially expressed genes between different samples were analyzed (40 (link)). Due to a large amount of transcriptome differential analysis data, Benjamini–Hochberg adjustment was performed on p-value to further reduce the false-positive rate. Subsequently, we defined genes with more than a 2-fold difference and p < 0.001 after correction as significantly differentially expressed genes.

Pericarp tissues, including the flavedo and the albedo tissues (three biological replicates each for WT and MT), were used for extracting total RNAs as described by Ma et al.^{52 (link)}. The cDNA library was constructed and the products were qualified, quantified, and sequenced using an Agilent 2100 Bioanaylzer, an ABI StepOnePlus Real-Time PCR System, and an Illumina HiSeqTM2000, respectively.

Transcriptomic profiling was analysed using cucumber fruits from WT and sf1 at 6 DBA and from the F2 population from sf1 × ‘Chinese long’ 9930 at 2 DBA and 9 DAA. The middle part of the fruits at all three stages was used to extract total RNA. The F2 population from sf1 × ‘Chinese long’ 9930 was used at the two later time points to test the fine mapping results obtained through SNP association analysis. Thirty individual plants were used for fruit sampling; these fruits were then pooled as one biological sample for WT and mutant phenotyping. mRNA was isolated from total RNA using oligo-dT magnetic beads and fragmented with fragmentation buffer for cDNA synthesis. After the addition of adenine, the resulting cDNAs were linked to adapters and purified by gel electrophoresis; finally, cDNAs approximately 250 bp in length were collected for PCR amplification. After quality checking and quantification with an Agilent 2100 Bioanalyzer and the ABI StepOnePlus Real-Time PCR System, all libraries were sequenced on the Illumina HiSeqTM 2000 platform at Honortech (Beijing, China).

After purification with oligo (dT)₂₅-attached magnetic beads, the mRNA was interrupted into short fragments by divalent cations under elevated temperature. Then, these cleaved RNA fragments were used to synthesize first-strand cDNA using a random hexamer primer and the SuperScript III (Invitrogen, USA) reverse transcriptase. The second-strand cDNA was subsequently synthesized using random primers and end repaired, then adaptors were ligated by T4 DNA ligase after adenylation at the 3′-end. Eventually, suitable adaptor-ligated fragments were selected as templates for PCR amplification to generate the final cDNA library. The four resulting cDNA libraries were quantified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and qRT-PCR (ABI StepOnePlus Real-Time PCR System, USA), and then RNA Sequencing (RNA-seq) was carried out with an Illumina HiSeq™ 2000 system at The Beijing Genomics Institute (BGI, Shenzhen, China). The entire set of raw reads was submitted to NCBI Sequence Read Archive under the accession number: PRJNA565345.

Total RNA was first isolated from samples, and then small RNAs (18–30 nucleotides) were recovered by PAGE and subsequently ligated with 3′ and 5′ adaptors. A small-RNA library was established after reverse transcription, PCR amplification and purification of the RNA fragments with adaptors. An Agilent 2100 Bioanalyzer and an ABI StepOnePlus Real-Time PCR System were used for quality and quantity detection, respectively, and an Illumina HiSeq 2000 sequencing system was used for sequencing of the generated small-RNA library.
High-quality clean full-length tags were obtained through quality control (QC) and filtration of raw data. The extracted clean reads were mapped to the tea tree genome (http://www.plantkingdomgdb.com/tea_tree/) using Bowtie2. Then, the clean tags were aligned with known miRNA sequences from miRBase (http://www.mirbase.org/) and matched with rRNAs, tRNAs, snRNAs, snoRNAs, repeats, exons, introns, and other elements. The nonannotated clean tags were used to predict novel miRNAs on the basis of the biological characteristics of miRNAs by MirDeep v0.2 (http://sourceforge.net/projects/mireap/files/latest/download). The DEMs were filtered with criteria of a fold change ≥ 2 and an FDR ≤ 0.01 by DEGseq v1.18.0 and DESeq2 v1.4.5. Then, psRobot v1.2 and TargetFinder v1.0 were employed to predict miRNA target genes.

Total bacterial DNA of rat fecal samples was extracted using a HiPure Stool DNA Kit according to the manufacturer’s instructions. The 16S rRNA V3–V4 region was amplified by PCR (98°C for 30 s, followed by 30 cycles at 98°C for 10 s, 60°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 120 s) using the following primers: 341F: 5′-CCTACGGGNGGCWGCAG-3′ and 806R: 5′-GGACTACHVGGGTATCTAAT-3′. The generated amplicons were purified using the AxyPrep DNA Gel Extraction Kit and quantified using the ABI StepOnePlus Real-Time PCR System before sequencing on the Illumina platform. Raw reads of all samples were uploaded to the NCBI Sequence Read Archive database (Accession No. PRJNA835243).
Bioinformatic analysis (OTUs, community composition, indicator species, α-diversity, and β-diversity) of the raw data was performed using various software tools, including Userach (version 7.0), Krona (version 2.6), Vegan (version 2.5.3), QIIME (version 1.9.1), and Muscle (version 3.8.31).