GSM8532574: StitchR 3.0 GFP Nt-GFP + Ct-GFP Biological Replicate 2; Homo sapiens; OTHER
The total RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA). TruSeq Stranded Total Sample Preparation Kit (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer's protocols. Briefly, ribosomal-depletion was performed on 200ng total RNA with biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads, followed by RNA fragmentation. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis using dUTP incorporation for strand marking. End repair and 3` adenylation was then performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA and amplified with PCR primers specific to the adaptor sequences to generate cDNA amplicons of approximately 200-500bp in size. The amplified libraries were sequenced using the NovaSeq 6000 (Illumina, San Diego, CA).