GSM6719235: GAT_56; Mus musculus; ncRNA-Seq
After dissection, organs were frozen on dry ice. Total RNA was isolated with the miRNeasy Micro kit (Qiagen). RNA quantity and quality were assessed by Agilent 2100 Bioanalyzer (Agilent Technologies) on randomized samples. Small RNA library preparation was performed using the MGIEasy Small RNA Library Prep Kit and the high-throughput MGI SP-960 sample prep system according to the manufacturer's protocols. In brief, 3'- and 5'-adapters were ligated to the RNA, and reverse transcription (RT) was performed using an RT primer, in which sample-specific barcodes were incorporated. The resulting cDNA was amplified in a polymerase chain reaction (PCR) with 21 cycles. The amplification product was size selected and purified using AMPure Beads XP (Beckman Coulter, Germany). The size of the purified PCR products was checked using an Agilent DNA 1000 Kit (Agilent Technologies), and the concentration was determined using Qubit™ 1X dsDNA High Sensitivity (HS) (Thermo Fisher Scientific). For each library, 16 samples, barcoded with barcodes 1-4, 13-16 and 25-32, were pooled in an equimolar fashion at a concentration of 4.56 ng/µl. Pooled libraries were circularized and sent for sequencing. A total of 65 libraries consisting of 947 samples were analysed in the project.