GSM5069465: US-1635402; Homo sapiens; RNA-Seq
RNAseq cDNA library was prepared with TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat Sample Prep Kit (Illumina, #RS-122-2203). Starting with 100 ng of total RNA, cytoplasmic ribosomal RNA was removed by hybridization to a biotinylated probe selective for ribosomal RNA species, followed by streptavidin bead binding and sample purification.  The resulting cytoplasmic rRNA-depleted sample was chemically fragmented and converted into single-stranded cDNA using reverse transcriptase and random hexamer primers, with the addition of Actinomycin D to suppress DNA-dependent synthesis of the second strand.  Double-stranded cDNA was created by removing the RNA template and synthesizing the second strand in the presence of dUTP in place of dTTP.  A single A base was added to the 3' end to facilitate ligation of sequencing adapters, which contain a single T base overhang.  Adapter-ligated cDNA was amplified by polymerase chain reaction to increase the amount of sequence-ready library.  During this amplification the polymerase stalls when it encounters a U base, rendering the second strand a poor template.  Accordingly, amplified material used the first strand as a template, thereby preserving the strand information.  Final cDNA libraries were analyzed for size distribution using the Agilent Tapestation 2200 (D1000 kit, Agilent # 5067-5582 and 5067-5583), quantitated by qPCR (KAPA Library Quant Kit, KAPA Biosystems # KK4824), then normalized to 2 nM in preparation for sequencing. Prepared libraries were sequenced with 2 × 50 bp paired-end reads on the Illumina HiSeq 2500 sequencer with a minimum of 3 GB of raw RNA sequencing data (Illumina, San Diego, USA). TruSeq Stranded Total RNA Ribo-Zero library preparation