Hiseq 4000 sbs kit

Seven-day-old mycelia of the WT and ΔUvCGBP1-33 strains were collected from PSB culture and used for total RNA isolation. RNA-seq libraries were created using an Illumina TruSeq RNA Sample Preparation Kit and then sequenced on an Illumina HiSeq 4000 system (IGENEBOOK Biotechnology Co., Ltd, Wuhan, China) based on HiSeq 4000 SBS Kit (300 cycles) protocol. Three biological replicates were carried out for each strain. The raw data of paired-end reads were filtered by SeqPrep and Sickle with default parameters. The software Tophat (version 2.0.14) and Cufflinks (version 2.2.1) were used to map clean reads to the U. virens genome and to calculate differential expression [32 (link)]. Filter conditions fold change >2 and adjusted p < 0.05 were applied for identification of differentially expressed genes [33–35 (link)].

Total RNA from the liver of ten male mice (ND, n = 5; HFD, n = 5) was extracted using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. And high-throughput sequencing was performed in the liver tissues of each mouse individually (Majorbio BioPham Technology Co., Shanghai, China). Briefly, two small RNA libraries specifically from five ND-fed mice and five HFD-fed mice were generated using the Truseq™ small RNA sample preparation Kit (Illumina, San Diego, CA, USA), and the small RNA sequencing was performed using HiSeq 4000 SBS Kit (Illumina, San Diego, CA, USA).

Gene expression analysis of the MRSA ATCC 43300 strain was analyzed after treatment with diclofenac (62.5 µg mL⁻¹) or 0.1% DMSO. Samples were collected after 8 h of treatment, and three independently prepared RNA samples from each condition were used for RNA Sequencing. Illumina sequencing was performed by Shanghai Majorbio Bio‐pharm Technology Co., Ltd. (Shanghai, China) using the Illumina TruSeq RNA sample prep kit and HiSeq 4000 SBS kit (Illumina, Inc.). After sequencing, the data were analyzed using edgeR software, and statistical significance was defined at p < 0.05. Only genes that were significantly differentially regulated (p < 0.05) by at least two‐fold compared to the control were considered.

S. aureus were cultured with Ti6 and HA/MoS₂–Ti6 for 6 h and then S. aureus were collected to extract the total RNA using TRIzol reagent (Invitrogen, CA, USA). RNA sequencing was performed via the HiSeq 4000 SBS Kit (300 cycles; Illumina, CA, USA). Data analysis was performed by FastqStat.jar (v0.11.4) and RSeQC (v2.6.4). Gene Ontology (http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genome (http://www.genome.jp/kegg/) were used to analyze the gene functions. Differential gene expression analysis was performed using the R package edgeR (v3.24), and those genes conformed to |log2FC | > 1 (p-value < 0.05) were considered to be differentially expressed genes.

Total RNA from mixed tissues of MP carapace (mixture of 5 individuals, body weight 86–100 mg, stage I and stage II) and EP carapace (mixture of 3 individuals, body weight 260–300 mg, stage IV), was extracted using Trizol Reagent (Invitrogen), followed by Illumina sequencing. Briefly, mRNA with poly (A) was isolated from total RNA using Oligo (dT) beads (Invitrogen). The mRNA was broken into short fragments (about 200 bp) using fragmentation buffer. These fragments were used as templates to synthesize the first-strand cDNA with random hexamers, after which a second-strand cDNA was synthesized. Adaptors were ligated onto the second-strand cDNA following by Illumina Hiseq sequencing (HiSeq 4000 SBS Kit (300 cycles), Illumina). The raw reads were quality controlled using Trimmomatic to generate clean reads, before performing de novo assembly through Trinity (v2.5.1). All clean reads were aligned with Bowtie2 (v2.3.4), followed by joint abundance estimation and RSEM to calculate transcripts per million (TPM) values.

Total genomic DNA was extracted from nine collected samples using the E.Z.N.A.^® DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. Concentration and purity of extracted DNA were determined with TBS-380 and NanoDrop2000, respectively. DNA extract quality was checked on 1% agarose gel.
DNA extract was fragmented to an average size of about 300 bp using Covaris M220 (Gene Company Limited, Hong Kong, China) for paired-end library construction. A paired-end library was constructed using a TruSeq^TM DNA Sample Prep Kit (Illumina, San Diego, CA, USA). Adapters containing the full complement of sequencing primer hybridization sites were ligated to the blunt ends of fragments. Paired-end sequencing was performed on an Illumina HiSeq4000 platform (Illumina Inc., San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using a HiSeq 4000 PE Cluster Kit and HiSeq 4000 SBS Kit according to the manufacturer’s instructions (www.illumina.com).