GSM5643290: Sample 43 Illumina; Homo sapiens; RNA-Seq
Three milliliters of whole blood was collected from an antecubital vein using the Tempus Blood RNA tubes (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was isolated from the whole blood according to the manufacturer's instructions (Tempus Spin RNA Isolation Kit, Thermo Fisher Scientific, Waltham, MA, USA). The purified total RNA was eluted in 90 μL elution buffer and stored in three aliquots at −80°C until further analysis. Five hundred nanograms of total RNA was used for sequencing according to the Illumina TruSeq Stranded mRNA sample prep guide - high sample protocol (Illumina, San Diego, CA, USA). Briefly, mRNA molecules were purified using the poly-T oligo attached magnetic beads following which the mRNA was fragmented and primed for cDNA synthesis. A single “A” base was subsequently added to the 3-prime end of the synthesised blunt-ended cDNA and ligated with index adapters for hybridisation onto a flow cell. The DNA fragments with adapters on both ends were amplified via polymerase chain reaction to generate the final double-stranded cDNA (ds-cDNA) library followed by library validation and normalisation and pooling of the samples. Samples were sequenced at 2x75 bp read length to a depth aimed at approximately 64 M reads per sample on the Illumina NextSeq 500 (Illumina, San Diego, CA, USA). Four hundred nanograms of total RNA was used for sequencing on the MGI DNBSEQ-G400RS instrument (MGI, Shenzhen, China). Total RNA was first treated with Globin-Zero Gold Kit (Illumina, San Diego, CA, USA) for rRNA depletion and globin mRNA reduction. The ds-cDNA library preparation is in line with the Illumina RNA-seq protocol described above. The ds-cDNAs were then heat denatured and circularised by the splint oligo sequence to generate the single strand circle DNA followed by rolling circle replication to create DNA nanoballs (DNB) for processing on the MGI DNBSEQ-G400RS.