GSM9046839: 63c0dc2eee730a0c;MVA-NP+M1; Homo sapiens; RNA-Seq
Human total RNA from blood (collected in PAXgene tubes, http://www.bdbiosciences.com/us/applications/blood-collection/cell-biomarker-preservation/paxgenereg-blood-rna-tube/p/762165) was extracted using the Agencourt RNAdvance blood kit (Beckman Coulter) on an automated Biomek Fxp workstation (Beckman Coulter).The purity of the RNA was assessed using a NanoDrop (Thermo Fisher Scientific) and quantified with the Quant-iT RiboGreen RNA Assay kit (Thermo Fisher Scientific). RNA integrity (RIN) was measured using the RNA Nano 6000 Assay kit on an Agilent Bioanalyzer 2100 system (Agilent Technologies) and the samples with RIN above 7.0 were processed to libraries. The mRNA libraries were prepared using the Universal Plus mRNA-Seq Library preparation kit (NuGEN) on an automated Biomek i7 workstation (Beckman Coulter). In brief, mRNA was purified from 400 ng of total RNA using oligo-dT beads, and cDNA was synthesized according to NuGEN's protocol. The AnyDeplete kit (NuGEN) was used to remove ribosomal RNA (rRNA) and globin transcripts before library amplification. The purified libraries were quantified with the Qubit dsDNA HS kit (Thermo Fisher Scientific), and their size and distribution were analyzed using a Bioanalyzer with the High Sensitivity DNA kit (Agilent). After library preparation, samples were pooled, and library integrity was verified by sequencing on an Illumina MiSeq. Deep sequencing was then conducted on a NovaSeq 6000 (Illumina, Inc., San Diego, CA), with an average depth of 30 million 100 bp paired-end reads per sample.