Truseq small rna sample prep kit

The mirVana miRNA Isolation Kit (Ambion) and TruSeq Small RNA Sample Prep Kits were used to extract total RNA and construct the cDNA libraries. The whole process was carried out in strict accordance with the reagent instructions. First, T4 ligase was used to ligate a 5′ adapter and a 3′ adapter to the RNA molecules. Then a SuperScript II Reverse Transcription Kit (Invitrogen) was used to reverse‐transcribed 5′ and 3′ adapter‐ligated RNA to cDNA and PCR amplification was performed. Finally, the cDNA product was purified by RNA Gel Electrophoresis and gel recovery. The size and purity of the sample were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara). The Illumina sequencing platform was used for sequencing analysis.
Raw reads of fastq format were processed consisting of removing adapter and low‐quality reads including sequences with quality score less than 20 and sequences with N base to obtain high‐quality clean reads. The Bowtie2 was use to map the clean reads to mature miRNAs in miRBase 21.0 database. These consistent sequences were considered as the known miRNAs. The expression level of miRNAs was measured by TPM. The p value was calculated by Audic–Claverie statistic. The fold change ≥ 2 or fold change ≤ 0.5, and p value < 0.05 were used as the cut‐off criteria.

After total RNA was extracted from the samples, a miRNA sequencing library was constructed using the mirVana miRNA Isolation Kit (Ambion) and TruSeq Small RNA Sample Prep Kits. Total RNA, concentration and integrity were measured by Nanodrop 2000 and Agilent 2100. After PCR amplification and quality inspection, the library was sequenced using an Illumina HiSeq X Ten platform. High-quality clean reads were obtained after filtering using Fastx-toolkit (0.0.13) software, Bowtie (1.1.1) and NGSQCToolkit.
The clean reads were aligned to the mouse genome and compared with miRBase (version 22.0). Target genes of DE miRNAs were predicted by Miranda (3.3a). The expression levels of the identified known mature miRNA sequences and the newly predicted miRNAs were quantified as the transcripts per million (TPM). DE miRNAs were analyzed with DESeq2 (1.18.0) software. The p values were calculated with the Audic–Claverie statistic. A fold change ≥2 or ≤ 0.5 and a p value < 0.05 were used as the cutoff criteria.

The circRNA and mRNA library were constructed using the TruSeqTM Stranded Total RNA Kit (Illumina, San Diego, CA). And the ribosomal RNA (rRNA) was removed from 2 μg total RNA by the Ribo-Zero Magnetic kit (EpiCentre Biotechnologies, Madison, WI, USA). The RNA was fragmented and reverse-transformed to synthesize cDNA, then the adaptor was ligated. The cDNA second strand was digested with UNG enzyme, amplified by polymerase chain reaction (PCR), and purified to obtain the final library. The miRNA library was constructed using the TruSeqTM Small RNA sample prep Kit (Invitrogen) according to the instructions. After removing the rRNA, the 3' and 5' end adapters were connected to the kit separately, and then the primers were reversed and PCR cycles were performed. Next, the library was enriched, purified and quantified [27 ]. At last, high-throughput sequencing was conducted using the Illumina NovaSeq 6000 ( Supplementary Fig. 1).