GSM2486459: KOPN-8 D28 Ctrl rep2; Homo sapiens; RNA-Seq
Cells were pelleted, flash frozen, RNA extracted with AllPrep DNA/RNA Mini Kit. Total RNA samples were converted into cDNA libraries using the TruSeq Total Stranded RNA Library Prep Kit (Illumina) Starting with 100 ng of total RNA, polyadenylated RNA (primarily mRNA) was selected and purified using oligo-dT conjugated magnetic beads. This mRNA 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 and using an Agilent Bioanalyzer (DNA 1000 kit, Agilent # 5067-1504), quantitated by qPCR (KAPA Library Quant Kit, KAPA Biosystems # KK4824), then normalized to 2 nM in preparation for sequencing
GEO Accession
GSM2486459