title: GSM6341132: Ventral polyA-enriched 3; Rattus norvegicus; RNA-Seq
library_construction_protocol: Poly-A enriched data: total RNA was extracted using Qiazol reagent according to manufacturer's protocol. Ribosomal RNA depleted data: total RNA was extracted using ExtractRNA reagent (Evrogen, Russia) following manufacturer's protocol. Depletion of ribosomal RNA was performed using MGIEasy rRNA Depletion kit. 1) mRNA isolation from total RNA with oligo(dT)-coated magnetic beads; 2) RNA fragmentation; 3) cDNA generation by reverse transcription using random primers followed by double-stranded cDNA synthesis; 4) reparation of ends, 3'-adenylation, ligation of adapters; 5) polymerase chain reaction using adapters. PCR products were purified with Ampure XP Beads and dissolved in EB solution; 6) the library has been validated with an Agilent Technologies 2100 bioanalyzer; 7) the double-stranded PCR product was denatured and cyclic molecules were synthesized from it using the splint oligo sequence. The result was a library of single-stranded cyclic molecules; 8) the library was amplified with phi29 DNA polymerase to form nanodroplets containing more than 300 copies of a single DNA molecule