Rna nano 6000 analysis kit

Mammary gland tissues from 4 animals in WT and Nrf2(-/-) groups were randomly selected for RNA sequencing (n = 4). TRIzol reagent was used to extract total RNA from each sample (Invitrogen, CA, USA). RNA integrity was assessed with RNA Nano 6000 Analysis Kit in the Bioanalyzer 2100 System (Agilent Technologies, CA, USA). In total, 16 samples were qualified to transcriptome analysis. A total amount of 3 μg RNA per sample was used as input material for the RNA sequencing library preparations. The libraries were sequenced by the high-throughput Illumina sequencing platform (HiSeq 4000) of Novogene Bioinformatics Institute (Beijing, China) using standard procedures (https://www.novogene.com/tech/service/etwithref/product/).

Total RNA was extracted using the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions. DNase I (Promega, Madison, WI, USA) was used to clean up extracted RNA and remove contaminants. The concentration and integrity of the RNA samples were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA), RNA Nano 6000 analysis kit, and Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) to confirm that they complied with sequencing requirements. It was found that the extracted total RNA satisfied the quality standards and could therefore be utilized for the construction of small RNA libraries if 1.8 < OD260/OD280 < 2.0, RIN > 7.0, and rRNA 28S/18S ≥ 0.7. Utilizing the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (New England Biolabs, Ipswich, MA, USA), sequencing libraries were created. A Bioanalyzer 2100 (Agilent Technologies) was used to test the quality of the libraries. The miRNA libraries were sequenced using the HiSeq™ 2500 platform (Illumina, USA).