Hiseq 4000 pe cluster kit

The extraction of total RNA from 0 and 1.5 mM H₂O₂ concentrations was performed using the TRIzol® Reagent RNA Purification Kit for bacteria (Invitrogen) according to the manufacturer’s instructions. A Nanodrop 2000 spectrophotometer evaluated the integrity and the quality of the extracted total RNA. All RNA samples were DNAse treated using the Ribo-Zero Magnetic kit to eliminate every contaminating genomic rRNA. The experiments were performed in triplicate for each stress treatment. RNA sequencing was done on the Illumina HiSeq4000 according to the manufacturer’s protocol (Illumina Inc., Majorbio, China). Briefly, the quality and the integrity of the total RNA were evaluated using the Agilent 2100 Bioanalyzer. RNA with an RNA Integrity Number (RIN) of 7.0 or higher was used for sequencing library preparation and processing. Sequencing libraries were adjusted using the TruSeq Stranded Specific mRNA Library sample Prep Kit as per the manufacturer’s guidelines (Illumina). Cluster generation was done according to the manufacturer’s references for onboard clustering (HiSeq 4000 PE Cluster Kit, Illumina). Next, sequencing (2 × 150 bp) was performed using Illumina Hiseq 300 platform.

Total RNA was extracted using Biozol reagent (Bioer, Hangzhou, China), and was then visualized on 1% agarose gel. The RNA quality was estimated using a NanoPhotometer^® spectrophotometer (IMPLEN, CA, USA) and the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA samples with an RNA integrity number (RIN) higher than 8.0 were qualified. The RNA quantity was determined using the Qubit^® RNA assay kit (Life Technologies, CA, USA).
In order to construct sequencing libraries, mRNA was enriched by treating total RNA with the Epicentre Ribo-zeroTM rRNA removal kit (Epicentre, USA). Sequencing libraries were constructed using NEBNext^® Ultra^TM directional RNA library prep kit for Illumina^® (NEB, USA), according to the manufacturer’s instructions. Next, DNA fragments were purified with AMPure XP (Beckman Coulter, Beverly, USA) and treated with 3 μl of USER enzyme (NEB, USA) at 37 °C for 15 min. DNA fragments were then amplified using Phusion High-Fidelity DNA polymerase, universal PCR primers, and index (X) primers. Finally, PCR products were purified using AMPure XP and their quality was evaluated using the Agilent Bioanalyzer 2100.
Index-coded samples were clustered on a cBot cluster generation system with a HiSeq. 4000 PE cluster kit (Illumina). Afterwards, DNA libraries were sequenced on the Illumina Hiseq 4000 platform.

RNA was isolated from the lung tissue of different groups. The Implant NanoPhotometer® spectrophotometer detected the purity of RNA. According to the manufacturer's instructions, sequencing libraries were developed using the RNA Library Prep Kit for Illumina® (NEBNext® Ultra, NEB, USA). The AMPure XP system purified the PCR products, and the library quality was checked using the Agilent Bioanalyzer 2100 system. The HiSeq 4000 PE Cluster Kit (Illumina, USA) was performed to cluster the index-coded samples following the manufacturer's protocols. Finally, the library preparations were sequenced on a HiSeq 4000 platform for 150 cycles. All library construction and sequencing steps were performed at Shanghai Life Gene Technology Co., Ltd. The DEGseq R package was used for differential expression analysis of two groups. Genes with a log2 fold change of >0.58 and P < 0.05 were considered differentially expressed genes (DEGs). GO enrichment analysis of DEGs was performed using the DAVID database.