GSM5699580: PE02 [miRNA-seq]; Mus musculus; miRNA-Seq
Total RNA from sample was extracted by using RNAqueousTM-Micro Total RNA Isolation Kit (Ambion, AM1931) following manufacturer's instructions. [RNA-seq] 2.5 ng of total RNA were used to amplify the cDNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech-Takara). 1 ng of amplified cDNA was used to generate barcoded libraries using the Nextera XT DNA library preparation kit (Illumina). Basically, cDNA is fragmented and adapters are added in a single reaction followed by an amplification and clean up. The size of the libraries was checked using the Agilent 2100 Bioanalyzer High Sensitivity DNA chip and their concentration was determined using the Qubit® fluorometer (ThermoFisher Scientific). Libraries were sequenced on a HiSeq 2500 (Illumina) and processed with RTA v1.18.66.3. FastQ files for each sample were obtained using bcl2fastq v2.20.0.422 software (Illumina). [miRNA-seq] 500 pg of total RNA were used to generate barcoded miRNA-seq libraries using the Bioo NEXTflex Small RNA (BiooScientific). Briefly, 3´ and 5´ SR adapters were first ligated to the RNA sample. Next, reverse transcription followed by PCR amplification was used to enrich cDNA fragments with adapters at both ends. Adapter-ligated cDNA fragments from different samples were pooled and run in a 6% polyacrylamide gel. The 147 nt band, corresponding to the pooled miRNA libraries, was purified from the gel. Finally, the quantity and quality of the pooled miRNA libraries were determined using the Agilent 2100 Bioanalyzer High Sensitivity DNA chip. Libraries were sequenced on a HiSeq 2500 (Illumina) and processed with RTA v1.18.66.3. FastQ files for each sample were obtained using bcl2fastq v2.20.0.422 software (Illumina).