Agilent dna 1000 chips reagents

The samples were placed in 1 ml TRIzol (15596018, Invitrogen, USA) under liquid nitrogen. Total RNA was separated by phenol‐chloroform (P120617, Aladdin, China) extraction. Then, the RNA was treated with DNase I (18047019, Invitrogen, USA). Subsequently, a spectrophotometer (Nanodrop 1000, Thermo Scientific, USA) was used to measure the purity (OD260/OD280) of separated RNA (resuspended in 50 μl RNase-free water). Finally, a fluorimeter (Qubit 2.0, Thermo Scientific, USA) was used to quantify the accurate concentration of total RNA, while a bioanalyzer (Agilent DNA 1000 chips/Reagents, Agilent Technologies, USA) was used to assess the quality of recovered RNA to ensure that the recovered total RNA was of sufficient quality for preparing sequencing libraries.

miRNA library was prepared by using a Small RNA Sample Prep Kit (RS-200-0012, Illumina, USA). cDNA was converted from total RNA followed by being amplified by polymerase chain reaction (PCR). Then, the concentration of the miRNA library was quantified by using a fluorimeter (Qubit 2.0, Thermo Scientific, USA). Next, we diluted the miRNA library to 1 ng/μl. Subsequently, a bioanalyzer (Agilent DNA 1000 chips/Reagents, Agilent Technologies, USA) was used to measure the insert size of the miRNA library. And qRT-PCR was used to detect the effective concentration of the miRNA library. The effective concentration of miRNA library > 2 nM was considered satisfactory. Deep sequencing was performed on HiSeq 2500 (Illumina HiSeq 2500, Illumina, USA).