GSM7566857: HeLaK_GFPOE_N1; Homo sapiens; RNA-Seq
Total RNA was extracted using the RNeasy Mini Kit (QIAGEN) with on column DNase digest (RNase-Free DNase Set, QIAGEN) according to the manufacturer's instructions. Sequencing library contruction was perfomred at Biomarker (Münster, Germany) by using Hieff NGS™ Ultima Dual-mode mRNA Library Prep Kit. Briefly, purity, concentration and integrity of RNA sample were examined by NanoDrop, Qubit 2.0 and Agilent 2100 once the RNA samples were received. Only RNA with RIN value greater than 8 was used for library construction. The procedures are described as follow: first, mRNA was isolated by Oligo(dT)-attached magnetic beads, followed by random fragmention in fragmentation buffer. Then first-strand cDNA was synthesized with fragmented mRNA as template and random hexamers as primers, followed by second-strand synthesis with addition of PCR buffer, dNTPs, RNase H and DNA polymerase I. Subsequently, purification of cDNA was processed with AMPure XP beads. Later, double-strand cDNA was subjected to end repair, where Adenosine was added to the end and ligated to adapters. Also, AMPure XP beads were applied to select fragments within certain size range. Lastly, cDNA library was obtained by certain rounds of PCR on cDNA fragments generated from the previous step. In order to ensure the quality of library, Qubit 2.0 and Agilent 2100 were used to examine the concentration of cDNA and insert size. Q-PCR was processed to obtain a more accurate library concentration and assure adequate library concentration.