GSM5799818: PMAIonomycin_stimulated; Homo sapiens; RNA-Seq
Cells from each donor were labelled with sample tags, washed with stain buffer and pooled together in cold sample buffer to obtain ~25,000 cells for each of the unstimulated and stimulated samples. Two nanowell cartridges were primed and loaded with the pooled samples for 15min at room temperature on the BD Rhapsody Express instrument. Cell capture beads were prepared and loaded onto the cartridge. The cartridge was washed, cells were lysed, and beads were retrieved with mRNA molecules attached to every bead. The samples underwent reverse transcription and Exonuclease I treatment. cDNA was amplified using a custom immune panel via PCR. mRNA PCR products were separated from sample tag products with double-sided size selection using AMPure beads. Separated products were further amplified using PCR and then purified. PCR products were then PCR amplified using two index primers, one for unstimulated and one for stimulated. Libraries were diluted to 650pM and multiplexed for paired-end (2 x 115bp) sequencing on a NextSeq2000 Sequencing System (Illumina) at 200 cycles P3 for 1.1 billion reads.