Nanophotometer spectrophotometer

Total RNA was extracted from four cell lines, BxPC‐3‐LV‐TOB1, BxPC‐3‐LV‐NC, Patu8988t‐LV‐TOB1, and Patu8988t‐LV‐NC, using TRIzol^® Reagent (Invitrogen) according to the manufacturer's instructions. NanoPhotometer spectrophotometer and Agilent 2100 Bioanalyzer were applied to evaluate the quality of the RNA samples. The RNA‐Seq service was provided by Beijing Novogene. What tools used in this work contained Illumina HiSeq™ 4000 system (Illumina), the FastQC tool and the HISAT2 tool.

Human milk EV-RNAs were isolated from the remaining EV fraction with the miRNeasy Serum/Plasma Maxi kit using a 1:5 ratio of sample to Qiazol (Qiagen, Germantown, MD), then cleaned using the RNA Clean & Concentrator-5 Kit (Zymo Research, Irvine, CA). RNA quantity and quality was checked on a Implen NanoPhotometer spectrophotometer (München, Germany) and verified with the TapeStation 4200 total RNA ScreenTape chips (Agilent, Santa Clara, CA) prior to sequencing. As EV RNA is primarily small RNA with no intact ribosomal RNA present, we thus qualitatively examined our samples TapeStation traces. These traces were compared to other traces of milk EV miRNAs and were comparable to previous findings (data not shown).

Total RNA was extracted from the leaves of 12 cotton samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Three biological replicates were performed. The concentration, purity and integrity of RNA samples were detected by Qubit 2.0 Flurometer, NanoPhotometer spectrophotometer and Agilent 2100 bioanalyzer. The cDNA library construction and sequencing were performed by Wuhan Met Ware Biotechnology Co., Ltd. (www.metware.cn) using the Illumina high-throughput sequencing platform. Clean reads were obtained by referring to the method of (Chen et al., 2018 (link)), and mapped to the cotton reference genome (Gossypium hirsutum. L) using HISAT v2.1.0 (Kim et al., 2015 (link)). The gene alignment and the FPKM of each gene based on the gene length were calculated using Feature Counts v1.6.2 (Liao et al., 2014 (link)). The DEGs were analyzed using DESeq2 v1.22.1, which were identified according to |log2fold change|≥1 and false discovery rate (FDR)<0.05 (Love et al., 2014 (link)). And in order to infer the function of DEGs, BLAST software and KOBAS 2.0 software were used for KEGG pathway analysis.

H. pylori NCTC 11637 strain grouping was the same as Genome DNA analysis and each group were run using three replicates. Total RNA was extracted with RNAprep Pure Cell/Bacteria kit (DP430, TIANGEN, China) and quality control was assessed using agar gel electrophoresis, NanoPhotometer spectrophotometer, and Agilent 2100 bioanalyzer for integrity and purity. Eligible RNA sample was treated with Ribo-Zero Magnetic kit (Bacteria) (Epicentre, USA) to remove rRNA. The cDNA library was constructed according to the Strand-specific approach (31 (link)). For the quality of the cDNA library, Qubit2.0 Fluorometer was used for preliminary quantification, and then qRT-PCR was used for accurate quantification to ensure its effective concentration was above 2 nM. Library construction and sequencing were conducted by Novogene Tech (Beijing) Co., Ltd. Raw reads were filtered and mapped to the H. pylori NCTC 11637 genome using Bowtie2. Gene expression counts were quantified using HTSeq. Differentially expressed genes (DEGs) were analyzed using DESeq2. Genes with absolute log₂-fold changes >0.5 and multiple P-value <0.05 were considered DEGs.

The anthers collected at different stages were sent to Beijing Novogene bioinformatics technology Co., Ltd. for RNA-Seq. The purity and integrity of the RNA samples were determined by NanoPhotometer Spectrophotometer and Agilent 2100 Bioanalyzer. RNA integrity and possible contamination were further determined using 1% agarose gel electrophoresis. A total of 3 μg total RNA per sample was used in RNA-seq library preparation (each developmental stage with three biological replicates). Sequencing was done using an Illumina HiSeq 4000 sequencing platform with 150 base pair (bp) paired-end (PE) reads.

Total RNA was extracted from leaf and bulb tissues of C. yanhusuo using an RNA isolation kit (HuaYueYang Biotechnology, Beijing, China). RNA concentration, purity, and integrity were measured using a Qubit^® RNA Assay Kit in the Qubit^® 2.0 Fluorometer (Life Technologies, CA, USA), a NanoPhotometer^® spectrophotometer (CA, USA), and the Agilent Bioanalyzer 2100 system (CA, USA), respectively.

Total RNA was isolated from the nine testicular tissues with an RNA Nano 6000 Assay Kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s protocols. The Agilent 2100 bioanalyzer and NanoPhotometer spectrophotometer were adopted to assess RNA integrity and concentration.
Library preparation. Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly T oligo-attached magnetic beads, and cDNA was synthesized using mRNA as a template. Selected cDNA library fragments that were preferentially 370–420 bp in length were purified with the AMPure XP system (Beckman Coulter, Beverly, MA, USA). After PCR amplification, the PCR product was purified with AMPure XP beads, and the library was finally obtained. After construction, the library was initially quantified using a Qubit2.0 fluorometer. qRT-PCR was applied to accurately quantify the effective concentration of the library (higher than 2 nM) to ensure the quality of the library.
Transcriptomic sequencing. After the library was quantified, the different libraries were pooled according to the effective concentrations and the target amounts of data produced and sequenced with the Illumina NovaSeq 6000 machine with 150 bp ends read.