GSM8283790: liver, 6 month old ki mice, 1; Mus musculus; RNA-Seq
RNA was harvested using Qiazol reagent. All samples passed through the following steps of quality control (QC) before library construction: RNA purity measured by Nanodrop, tests RNA degradation and potential contamination using Agarose Gel Electrophoresis, RNA integrity checked by Agilent 21000. After QC procedures, mRNA was enriched using oligo(dT) beads. The mRNA was then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The final cDNA library was ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment. Library QC: Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then diluted to 1 ng/µl before checking insert size on an Agilent 2100 and quantifying to greater accuracy by quantitative PCR (library activity >2 nM).