GSM6398508: Uninfected_RK_2; Homo sapiens; RNA-Seq
Total RNA was extracted from virus containing cell lysates using Trizol LS (Invitrogen, Carlsbad, CA) and purified using a column purification method (Qiagen, Germany) in accordance with manufacturer's recommendations that were modified to isolate both small and large RNA species. RNA concentration and preliminary quality control was estimated on a NanoDrop spectrophotometer (ThermoFisher Scientific) while RNA-Seq was performed commercially (BGI Americas, San Jose, CA). Libraries were constructed by BGI. Briefly, RNA was QC'ed and mRNA enriched by oligo(dT) beads, followed by fragmentation and ds cDNA synthesis. Ds cDNA was end repaired, added an A, and adapter ligated followed by UDG degradation of dUTP-marked strand and PCR. This was followed by single strand separation and digestion of uncircularized DNA to obtain the final library which was QC estimated. Sequencing: Single-stranded cyclized DNA molecules were replicated via rolling cycle amplification, and a DNA nanoball (DNB) containing multiple copies of DNA was generated. DNBs were loaded into patterned nanoarrays and sequenced through combinatorial Probe-Anchor Synthesis (cPAS) on the DNBSEQ platform.