Rna seq library preparation kit

In this study, 9 small RNA libraries and 9 long RNA libraries were prepared with three replicates in each group. Briefly, total RNA was isolated and purified using Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's procedure. The amount of RNA and purity of each sample were quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed using Agilent 2100.Small RNA sequencing library preparation uses TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA). About 5 µg of total RNA was used to deplete ribosomal RNA according to the instructions of the Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, USA), and the remaining RNA fragments were reverse transcribed using an RNA-seq Library Preparation Kit (Illumina) to form the final cDNA. Finally, we performed the paired-end sequencing on an Illumina Hiseq4000 (LC Bio, Hangzhou, Zhejiang, China), following the vendor’s recommended protocol.

Total RNA from lung tissues was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the RNA was reverse-transcribed into cDNA using a PrimeScript^TM RT Reagent Kit (TaKaRa) according to the manufacturer’s instructions. A cDNA library was established using an RNA-Seq Library Preparation Kit (Illumina, CA, USA) following the manufacturer’s protocol and used for Illumina HiSeq sequencing. FastQC software was used for quality control of the original data, and Trimmomatic software (Illumina, CA, USA) was used to preprocess the original data to remove the ribosomal RNA sequences and other possible interfering contaminants. The clean reads were aligned to a reference genome (https://www.ncbi.nlm.nih.gov/genome/?term=Macaque+nemestrina, (accessed on 1 March 2018)). All the sequence data were submitted to the SRA database, and the accession numbers were SRR9050947 to SRR9050956. The criteria for differentially expressed genes (DEGs) were a false discovery rate (FDR) of 0.01 and a log2 (fold change) ≥ 2. The significantly enriched pathways of the DEGs were determined by using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, which was performed using KOBAS software. STRING online software was used to search for the protein–protein interaction (PPI) network of the proteins encoded by the DEGs [27 (link)].

RNA from chondrocytes treated with IL-1β or IL-1β + resveratrol was extracted with TRIzol by standard methods. Concentration of total RNA, RIN value, 28S/18S, and fragment size were detected with Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit). Agarose gel electrophoresis was used to detect RNA integrity. The cDNA library was established using the RNA-Seq Library Preparation Kit (Illumina) following the manufacturer’s protocol. Sequencing was performed on the Illumina HiSeq4000 sequencing platforms at Majorbio Biotech Co., Ltd.

S2 cells were harvested and pelleted. 1 ml of TRIzol reagent was added per 10⁶ cells and pipetted to homogenize the cell lysate. RNA precipitated using isopropanol, dissolved in DEPC treated TE buffer and checked for quality before proceeding for library preparation using Illumina RNA seq library preparation kit.

In accordance with the manufacturer’s instructions, we used the Trizol reagent (Invitrogen, Carlsbad, CA, USA) to extract the total RNA from the mammary tissue. To remove genomic DNA from each RNA sample, we used DNase I (Takara, Dalian, Liaoning, China). Only RNA samples with suitable RNA electrophoresis results (28S/18S ≥ 1.0) and RNA integrity number (RIN) ≥ 7.5 could be analyzed further. In each experimental group, we used 5 μg of each RNA sample and mixed in pairs to prepare a total of three RNA pools. A Ribo-Zero^TM rRNA Removal Kit (Illumina, San Diego, CA, USA) was used to remove the ribosomal RNA from the RNA libraries, and the remaining RNA fragments were reverse transcribed using an RNA-seq Library Preparation Kit (Illumina) to form the final cDNA. Finally, we used an Illumina Hiseq 4000 sequencer (LC Bio, Hangzhou, Zhejiang, China) for paired-end sequencing of the libraries.

We used TRIzol (Invitrogen) to extract total RNA from blood-derived exosomes, as per the manufacturer’s instructions. To remove genomic DNA, RNA samples were treated with DNase I (Takara, Dalian, China). RNA samples with RNA integrity number ≥ 7.5 were further analyzed. In each experimental group, two randomly selected samples were mixed in equal quantities to derive one sequenced pool. The Ribo-Zero^TM rRNA Removal Kit (Illumina, San Diego, CA, USA) was used to remove rRNA from RNA libraries, and the remaining RNA fragments were reverse transcribed to cDNA using the RNA-seq Library Preparation Kit (Illumina). Finally, paired-end sequencing of the libraries was performed on an Illumina HiSeq™ 4000 sequencer (LC Bio, Hangzhou, China).