GSM7695808: Kidney, Cisplatin-4; Mus musculus; RNA-Seq
Total RNA was extracted from each tissue sample using the Trizol method. After incubation of RNA with a biotin-modified probe, the probe was captured by a streptavidin magnetic bead to eliminate rRNA. The obtained samples were purified by AMPure XP magnetic beads, and then buffer composed of elution, fragmented, and random primers was added and the samples were incubated at 94°C. Thermal fracture led to fragments distributed between 100 and 300 bp. Using first-strand synthesis buffer and Invitrogen SuperScript IV reverse transcriptase, this fragmented RNA was used as a template to synthesize the first-strand cDNA. Actinomycin D was added to the first-strand synthesis buffer to prevent the reverse transcription of DNA templates and to ensure the reverse transcription specificity of RNA templates. After that, the double-stranded synthesis premix system was added to the synthesized first-strand cDNA, and the product was purified by Agencourt AMpure XP magnetic beads. From this reaction we obtained purified double-stranded cDNA and then added a 3' end plus "A" buffer reaction system to this system. Finally, ligation buffer and a double-stranded sequencing linker was added to the library; we used T4 DNA ligase to attach the Illumina sequencing linker to the DNA library. The library was purified using the Agencourt SPRI select Nucleic Acid Fragment Screening kit.