GSM9035652: Ctrl_2_1; Homo sapiens; RNA-Seq
We employed four parallel wells for each group of HK-2 cells: HK-2 cells transfected with the control lentivirus were cultured in a high-glucose medium containing 30 mM glucose (HG-NC) for 72 hours, while a separate group of control lentivirus-transfected HK-2 cells were cultured in a medium containing 30 mM mannitol to serve as an osmotic control (OS-NC). Similarly, HK-2 cells transfected to overexpress hnRNPF-lentivirus were maintained in a 30mM glucose medium (HG-OE) and in a mannitol medium (OS-OE) for 72 hours, respectively. All RNA was treated to be processed with RQ1 DNase (Promega) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260nm/280nm (A260/A280) utilizing smartspec plus (BioRad). RNA integrity was further verified by 1.5% agarose gel electrophoresis. For each sample, 1 μg of total RNA was used for RNA-seq library preparation. mRNAs were captured by VAHTS mRNA capture Beads (Vazyme, N401).The purified RNA was treated with RQ1 DNase (Promega) to remove DNA before used for directional VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (NR605) Polyadenylated mRNAs were purified and fragmented. Fragmented mRNAs were converted into double strand cDNA. Following end repair and A tailing, the DNAs were ligated to Adaptor (N323). After purification of ligation product and size fractioning to 300-500bps, the ligated products were amplified and purified, then quantified and stored at -80℃ before sequencing. The strand marked with dUTP (the 2nd cDNA strand) is not amplified, allowing strand-specific sequencing. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina Novaseq 6000 system for 150 nt paired-end sequencing.