title: GSM2881396: hsa.Akron5.Hela.3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted with Trizol (Ambion) For Akron-SMRT, total RNA was extracted by Trizol from HeLa total cells and sucrose gradient polysome fractions. RNA was treated with 5'-phosphate specific Terminator exonuclease to remove non-capped mRNA. The 3' ends of mRNA molecules were marked by ligation to an adapter that contains a biotin group at the 3' end, allowing for further purification. We generated cDNA in conditions that favor full-length molecule amplification using primers designed to specifically amplify mRNAs encoded by CDK4 and TUBB genes. To avoid capturing 5' OH mRNAs we used two PCR amplification steps for each gene using forward primers that started at the 5' UTR and at the start codon. We then size-selected by gel purification to enrich for larger mRNAs and performed SMRT sequencing.    For Akron3,  abundant small RNAs was first substracted by dynabeads size-selection, and ribosomal RNAs and small-capped RNAs (i.e U snRNAs) were depleted using synthesized biotinylated oligonucleotides.  Capped mRNAs were then  selected by Terminator exonuclease treatment followed by biotinylated adapter ligation at the 3' end and streptavidin capture. Similarly, for Akron5, polyadenylated mRNAs were enriched using Oligo(dT) followed by biotinylated adapter ligation at the 5' end and streptavidin capture. While capped mRNAs are carried over, only poly(A) mRNAs with 5' monophosphate are captured. The native ends of RNAs in Akron3 and Akron5 are marked by adapter ligation. In Akron3 and Akron5, RNA was chemically fragmented before the second adapter ligation. After fragmentation, RNA in Akron3 was size selected (>400 bp) on denaturing polyacrylamide. In Akron3 and Akron 5, a second adatpter was ligated and cDNA was generated by Superscript III with specific primers that recognize the adapter sequence. PCR amplification was performed with primers compatible with Illumina sequencer. PCR products were purified (~500-800 bp) on Metaphore agarose gels and were identified by paired-end, sequence-by-synthesis
attributes.tag: GEO Accession
attributes.value: GSM2881396