GSM5948784: 20_6046-T32_P25; Homo sapiens; RNA-Seq
Total RNA from PAXgene preserved blood was extracted using the Agencourt RNAdvance Blood Kit (Beckman Coulter) on a BioMek FXP Laboratory Automation Workstation (Beckman Coulter). Concentration and integrity (RIN) of isolated RNA were determined using Quant-iT™ RiboGreen™ RNA Assay Kit (Thermo Fisher) and an RNA Standard Sensitivity Kit (DNF-471, Agilent Technologies, Santa Clara, CA, USA) on a Fragment Analyzer Automated CE system (Agilent Technologies), respectively. cDNA libraries were constructed from total RNA using the Universal Plus mRNA-Seq kit (Tecan Genomics, San Carlos, CA, United States) in a Biomek i7 Automated Workstation (Beckman Coulter). Briefly, mRNA was isolated from purified 300 ng total RNA using oligo-dT beads and used to synthesize cDNA following the manufacturer's instructions. The transcripts for ribosomal RNA (rRNA) and globin were further depleted using the AnyDeplete kit (Tecan Genomics) prior to the amplification of libraries. Library concentration was assessed fluorometrically using the Qubit dsDNA HS Kit (Thermo Fisher), and quality was assessed with the Genomic DNA 50Kb Analysis Kit (DNF-467, Agilent Technologies). Following library preparation, samples were pooled, preliminary sequencing of cDNA libraries (average read depth of 90,000 reads) was performed using a MiSeq system (Illumina) to confirm library quality and concentration. Deep sequencing was subsequently performed using an S4 flow cell in a NovaSeq sequencing system (Illumina) (average read depth ~30 million pairs of 2×100 bp reads). In addition to the 8-base i5 and i7 barcodes, the i7 adaptor contains 8 random bases immediately following the library index. The additional 8 bases are used for duplicate read determination using the Duplicate Marking Tool. To take advantage of this feature, the libraries were sequenced using 16 cycles for the i7 index read.