GSM2152853: mRNA-associated with polysome in hCINAP overexpressed cells; Homo sapiens; RNA-Seq
Total RNA extraction was performed with the Qiagen RNeasy KIT. For the polysome RNA-seq, RNA associated with monosome or polysome was precipitated with 2 volumes of 8M guanidine-HCl and 3 volumes of 99% ethanol at -20°C overnight. For extraction of the RNA elutation, using the Qiagen RNeasy KIT. The RNA samples are first treated with DNase I to degrade any possible DNA contamination. Then the mRNA is enriched by using the oligo(dT) magnetic beads. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then the first strand of cDNA is synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with magnetic beads. End reparation and 3′-end single nucleotide A (adenine) addition is then performed. Finally, sequencing adaptors are ligated to the fragments. The fragments are enriched by PCR amplification. The integrity and concentration of the library were evaluated by Agilent Technologies 2100 Bioanalyzer and ABI StepOnePlusReal-Time PCR System, respectively.
GEO Accession
GSM2152853