GSM5828993: mice_9month-old-WT_rep3; Mus musculus; miRNA-Seq
RNA was extracted by Invitrogen's Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). following the manufacturer's recommendations. One microgram of total RNA was used for the construction of the cDNA library using the TruSeq Small RNA Sample Prep Kits (Illumina) according to the manufacturer's protocol. Next, the Illumina HiSeq 2500 at the LC-BIO (Hangzhou, China) was employed for the single-end sequencing according to the manufacturer's recommendations. Finally, the clean reads were obtained by Cutadapt V1.14 to remove the 3ʹ-adapter (5ʹ-TGG AAT TCT CGG GTG CCA AGG AAC TC-3ʹ) that controls the length between 17 and 35 bp. Trimmomatic V0.36 was used to delete the low-quality reads (Q < 20), and Blast V2.6.0 was used to remove the RNA families (ribosomal RNA, transfer RNA, small-nuclear RNA, and small-nucleolar RNA) and repeats using comparison conditions that were defined as a gap- open equal to zero, e-value less than 0.01, and mismatch less than 1. To identify novel miRNAs, miRbase V21.0 was used to screen for known miRNA to make a prediction. The unmapped sequences were further searched using miRDeep2 V2.0.0.8, and the mouse reference genome (http://asia.ensembl.org/ index.html) was used to distinguish novel miRNAs and predict their secondary structure (Friedländer et al., 2012).