Quantifluor st fluorometer

After fermentation of 24 h, high-throughput sequencing technology of bacterial 16S rRNA was implemented to investigate the impact of polysaccharides on the gut microbiota. Each sample of 24 h blank control fermentation group (CON group), 24 h inulin fermentation group (INU group), and 24 h Lyophyllum decastes (Fr.) Singer fermentation group (LDSPs group) was extracted using Qiagen QIAamp Fast DNA Stool Mini Kit, according to the manufacturer’s instructions. The DNA extraction from all samples was visualized on 1% agarose gel electrophoresis. PCR amplification was performed using TransStart FastPfu DNA Polymerase, and the amplicons were purified using the AxyPrep DNA gel extraction kit (Axygen Bioscience, Union City, United States) and quantified using QuantiFluor™-ST fluorometer (Promega, Madison, United States). The major PCR products from the V3–V4 region of the 16S rRNA gene amplified with primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R(5′-GGACTACHVGGGTWTCTAAT-3′) were sequenced on the Illumina Miseq platform by Shanghai Majorbio Bio-pharm Technology Co. Ltd. (Shanghai, China).

Fecal DNA was extracted from cecum stool samples using E.Z.N.A. soil kit (Omega Bio-Tek, GA, USA) following the manufacturer's instructions. The hypervariable V3-V4 region of the 16S-rDNA gene was amplified by PCR using the primers 338F: 5′-ACTCCTACGGGAGGCAGCAG-3′. The cycling conditions were as follows: initial denaturation at 95°C for 3 min, 28 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, elongation at 72°C for 45 s, and final extension at 72°C for 5 min and 10°C until completion. Amplicons were recovered from 2% agarose gels and purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA) following the manufacturer's instructions and quantified using a QuantiFluor-ST fluorometer (Promega, USA). The purified amplicons were pooled in equimolar ratio and paired-end sequenced (2 × 300 bp) using PE300 strategies on an Illumina MiSeq platform (Illumina, CA, USA). The raw reads were deposited into the NCBI Sequence Read Archive database (accession number: SRP168312).

The percentage of JAK2Δ14 compared to the full-length isoform JAK2+14 was calculated using absolute standard curves. The PCR products corresponding to the full-lenght transcript and skipped isoform (S2 Table) were run on 2% agarose gels in TBE buffer. The amplified fragments were excised and purified from the gel using QIAquick spin columns (Qiagen). The concentrations of the PCR products were measured using both the Quantifluor dsDNA System on a Quantifluor-ST fluorometer (Promega) and the Nanodrop 1000 spectrophotometer (Thermo Scientific). The molecular weight of the PCR fragments was determined using the software MacVector (MacVector, Inc.) and used for the conversion of micrograms to picomoles. Finally, equimolar dilutions of PCR fragments were used to generate the standard curves (S2 Fig.).

The bacterial genomic DNA was amplified with the 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 533R (5′-TTACCGCGGCTGCTGGCAC-3′) primers specific for the V1-V3 hypervariable regions of the 16S rRNA gene33 (link)34 (link)45 (link). Each forward primer incorporated FLX Titanium adapters and a sample barcode at the 5′ end of the reverse primer to allow all samples to be included in the single 454 FLX sequencing run. All PCR reactions were performed in 50-μl triplicates and combined after PCR. The products were extracted with the QIAquick Gel Extraction Kit (QIAGEN) and quantified on NanoDrop ND-1000 spectrophotometer, QuantiFluor-ST Fluorometer (Promega, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Equimolar concentrations of 83 samples were pooled and sequenced on a 454 Life Sciences Genome Sequencer FLX system (Roche, Basel, Switzerland) according to the manufacturer’s recommendations.

Poly (A) mRNA was isolated from 12 μg of total RNA extracted from approximately 1200 antennae of 3–4 days-old adult female moths using the PolyA+Tract mRNA Isolation System (Illumina, San Diego, CA), and further purified using the RNeasy MinElute Clean up Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Fragmentation buffer was added to cleave mRNA into short fragments, and then, these fragments were used to synthesize first-strand cDNA using random hexamer primers, which was further transformed into double stranded cDNA with RHase H and DNA polymerase I. A paired-end library was constructed from the cDNA synthesized using the Genomic Sample Prep Kit (Illumina). Fragments larger than 375 bp were purified with QIAquick PCR Extraction Kit (Qiagen), end-repaired, and linked with sequencing adapters. AMPureXP beads were used to remove the unsuitable fragments, and then, the sequencing library was constructed with PCR amplification. After being validated using Pico green staining (Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen, P7589) and fluorospectrophotometry, and quantified using Agilent 2100 (Quantifluor-ST fluorometer, Promega, E6090)), the library was sequenced using Illumina Miseq platform (Shanghai Personal Biotechnology Cp., Ltd. Shanghai, China). For subsequent analysis, 1/2 run data was generated.

mRNA with polyA tail was enriched by the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) using Oligo (dT) magnetic beads, and then mRNA was randomly interrupted by bivalent cations. Using fragmented mRNA as the template and random oligonucleotides as primers, cDNA was synthesized. The double-stranded cDNA was purified and repaired at the double end, and then the “A” base was introduced into the 3 ‘end to connect the sequencing connector. cDNA with a length of about 400–500 bp was screened by AMPure XP beads for further PCR amplification. The PCR product was purified by AMPure XP beads again, and finally the library was obtained. Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA, 2100) and Agilent High Sensitivity DNA Kit (Agilent, 5067–4626) were used for library quality detection. The total concentration of the library was detected using Pico green (Quantifluor-ST fluorometer, Promega, Madison, WI, USA; Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen, St. Louis, MO, USA). Finally, the concentration of the effective library was quantitatively detected by quantitative real-time polymerase chain reaction (qRT-PCR). Finally, using second-generation sequencing technology (Next-Generation Sequencing, NGS) and based on the Illumina sequencing platform, these libraries were sequenced according to the double terminal (Paired-end, PE).

DNA was extracted from the soil samples (0.4 g wet weight) with E.Z.N.A Soil DNA (OMEGA, United States) according to the manufacturer’s instructions. The V4 hypervariable region of bacterial 16S rRNA gene fragments were amplified in triplicate from each of the resulting DNA extracts using the primers 515F (5′-GTG CCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The amplification was carried out in 20 μL mixture 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase and 10 ng of template DNA. The amplification conditions involved an initial denaturing step at 95°C for 2 min followed by 25 cycles (95°C for 30 s, 56°C for 30 s, 72°C for 30 s) and a final extension at 72°C for 5 min.
Amplicons were purified using QIAquick PCR Purification Kit (Qiagen, China) and quantified using QuantiFluor-ST fluorometer (Promega, United States) according to the manufacturer’s instructions. Then the qualified libraries mentioned above were sequenced pair-end on the Illumina HiSeq System (Illumina, United States) by the sequencing strategy PE250.