GSM2157699: Control 1 PFC Illumina Hiseq miRNA; Mus musculus; miRNA-Seq
Twenty-four hours after the mice were surely tested to demonstrate CUMS-induced depression-like behaviors, these depression-like mice and controls were anesthetized by using Isoflurane and decapitated by guillotine. Both sides of the medial prefrontal cortex were quickly isolated and dissected on the ice-cold glass slides. These cortical tissues were placed into the frozen vials that contained RNAlater RNA Stabilization Reagent (QIAGEN, Germany) at 4°C and stored at -80°C for subsequent analysis. Total RNAs from the tissue of medial prefrontal cortex in each mouse were isolated with TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) based on the manufacturer instruction. Total RNA samples placed in the dried ice were delivered to Beijing Genomics Institute (BGI), China for sequencing analysis. RNA samples were done with quality-control by BGI staff using Agilent 2100 Bioanalyzer (Agilent Technologies, USA) with RNA 6000 nano Reagents Port 1. The concentration of total RNA, the value of RNA integrity number (RIN) and the ratio of 28S to 18S ribosomal RNA were measured. The samples with total RNA amount larger than 10 μg, the concentration larger than 200 ng/μl, the RIN larger than 8, and the ratio of 28S to 18S larger than 1.0 were selected for the construction of transcriptome and small RNA libraries, respectively. miRNA sequencing library was constructed from those total RNAs, in which low molecular weight RNAs (18-30 nt) were isolated by polyacrylamide gel electrophoresis. 5ˊ-RNA adapter was ligated to RNAs with T4 RNA ligase. The ligated RNA was size-fractionated and 36-50 nucleotide fractions were excised. 3ˊ-RNA adapter was subsequently ligated to the precipitated RNA by using T4 RNA ligase. The ligated RNAs were size-fractionated and the 62-75 nucleotide fraction (small RNA+adaptors) was excised. Small RNAs ligated with adaptors were subjected to RT-PCR to produce sufficient templates for sequencing. PCR products were purified and collected by gel purification and ready for high-throughput sequencing.