Steponeplus real time pcr system

All samples (n = 23) were homogenized in RLT Buffer (Qiagen, Valencia, CA) using a Bead Ruptor Mill Homogenizer (Omni International, Kennesaw, GA). Total RNA, including small and miRNA, was isolated using the MirVana miRNA Isolation Kit (ThermoFisher Scientific, Waltham, MA) following the manufacturer’s protocol. Total RNA concentration and 260/280 absorbance ratio were determined by the NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA) (Supplementary Table S1). The samples were processed using the TruSeq Small RNA Library Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. The following steps were included in small RNA library preparation: small RNA filtration on PAGE gel, adapter ligation, reverse transcription, PCR product purification, and library quality testing by the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and the ABI StepOnePlus Real-Time PCR System. Samples were then sequenced using the Illumina HiSeq 4000 (50 bp single-end) at the sequencing facility (BGI America, Cambridge, MA, USA) to reach the expected output of 10 million reads per sample.

Mouse kidneys and livers were lysed in TRIzol, and the total RNA was purified by chloroform extraction and isopropanol precipitation. RT was performed from 500 ng of total RNA with the Takara PrimeScript RT Reagent Kit (Ozyme, Saint Quentin en Yvelines, France). Primers and MGB-Taqman probes were purchased from ThermoFisher Scientific (Table S2). The PCR reaction mixture was prepared using the Brilliant II QPCR Master Mix (Agilent Technologies, Les Ulis, France). All PCR reactions were performed in a StepOnePlus Real-Time PCR System (Agilent Technologies, Thermo Fisher Scientific). The data were acquired and analyzed with the StepOne software (Agilent Technologies). Target gene expression was normalized on the basis of the GUSB content of each sample, and was subsequently normalized to a basal mRNA level with the equation N target = 2^{ΔCt sample}, where ΔCt is the Ct value of the target gene minus the Ct value of the HPRT gene. The results are reported as “normalized mRNA levels”—i.e., the N target value divided by the N target value of the smallest quantifiable amount of target gene mRNA (target gene Ct value = 35).

Assay for transposase-accessible chromatic with high-throughput sequencing (ATAC-seq) is a new technology for screening transcription factors [24 (link)]. This technology was performed by Annoroad Gene Technology, the protocol as following. Around 50,000 living cells were taken for each library preparation. The cells were lysed in 1 × lysis buffer to get the nuclei, and TruePrep™ DNA Library Prep Kit V2 for Illumina (Vazyme Biotech) was used to construct the transposase -treated libraries. The mass concentration and molar concentration of libraries were detected by Qubit 3.0 Fluorometer and StepOnePlus™ Real-Time PCR system, respectively, and lengths of inserted fragments were detect with Agilent HS 2100 Bioanalyzer. Qualified libraries were sequencing by IlluminaHiSeq X ten platform in pair-end 150 bp style.
Raw data was stored in FASTQ format, including the base sequence and corresponding quality information. Adaptor-polluted or low-quality reads were then filtered out to get the clean data. Clean data was mapped to reference genome by Bowtie2, and visualized by IGV (Integrative Genomics Viewer). Peaks corresponding to the open region in genome were detected by MACS2.