GSM7506625: LTT 1 (PA 8695); Papio anubis; RNA-Seq
RNA was isolated using the RNeasy minikit (Qiagen #74104)) and treated with Rnase-free Dnase (Ambion #AM1906) according to the manufacturer's insructions. Sequencing libraries were prepared using strand-specific QuantSeq 3' RNAseq kit (Lexogen, FWD, Cat. No. 015). The QuantSeq protocol generates only one fragment per transcript, resulting in accurate gene expression values, and the sequences obtained are close to the 3' end of the transcripts. Total RNA in the amount of 50 nanograms per sample was used as an input. Library construction was performed according to the manufacturer's protocol. In brief, during the first strand cDNA synthesis, an oligo dT primer containing an Illumina-compatible sequence at its 5' end was hybridized to mRNA, and reverse transcription was performed. After that, the RNA template was degraded, and during second strand synthesis, the library was converted to dsDNA. Second-strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5' end. The double-stranded libraries were purified by using magnetic beads to remove all reaction components. Next, the libraries were amplified to add the complete adapter sequences required for cluster generation and to generate sufficient material for quality control and sequencing. A number of PCR amplification cycles was 18, as determined by test qPCR using a small pre-amplification library aliquot for each individual sample.  Final amplified libraries were purified, quantified, and fragment sizes were confirmed to be within 275-300 bp by gel electrophoresis using Agilent 4200 TapeStation (D5000 Screen Tape). Concentration of the final library pool was confirmed by qPCR.