GSM5398958: POLY_FU10_S2; Homo sapiens; ncRNA-Seq
RNAs were extracted from the fractions containing 2 to 9 ribosomes per RNA. 150 µL of each fraction was collected and pooled and RNA were extracted using TRIzolTM LS reagent (Invitrogen) following manufacturer instruction and suspended in RNAse-free water. For sequencing, RNA samples were processed using Stranded mRNA Prep kit (Illumina) according to manufacturer's protocol. Briefly, mRNA molecules containing polyA tails were capture by oligo(dT) magnetic beads. Then, purified mRNA was fragmented and copied into first strand complimentary DNA (cDNA) using reverse transcriptase and random primers. In a second strand cDNA synthesis step, dUTP replaced dTTP to achieve strand specificity. In the final steps adenine and thymine bases were added to fragment ends and adapters were ligated. The resulting library was purified and selectively amplified for sequencing. Next generation sequencing was performed on NextSeq 500 System (Illumina) using high output 2x75bp Flowcell (Illumina).