GSM6280653: CPH303_LPMC_CD4; Homo sapiens; RNA-Seq
Stimulated LPMCs from three donors (141, 194 and 267) were washed with ice-cold BD stain buffer, resupended in stain buffer containing Fc-block for 10 min at RT and then washed again. Staining with with BD sample tags and a panel of 50 BD Abseq antibodies was performed in BD stain buffer on ice for 1 hour. Cells were washed with addtional staining buffer and resuspended in a Mastermix containing near-infraread fluorescent LIVE/DEAD dye and flurecently labelled antibodies (CD4, CD8, CD45, TCRgd, Va7.2, Va24-Ja18, TCRgd) for sorting. Live CD45+CD4+ cells negative for all other markers were sorted RPMI containing 20% FCS. After the sort, cells were washed and equal cell numbers form each sample (3 donors, 4 condtions = 12 samples) were pooled together to 20.000 cells and loaded onto a BD Rhapsody cartridge. Libraries for the targeted mRNA, sample Tags and Abseq were constructed using the BD Rhapsody Targeted mRNA and Abseq Amplification kit using the recommended reagents and manufacturer's instructions (Protocol Doc IDs: 210967 Rev. 1.0 and 214508 Rev. 3.0). Briefly, single cells were captured  with beads on a BD Rhapsody cartridge, lysed, beads were recovered , cDNA synthesis and an exonuclease I treatment were performed to obtain cDNA containing the information about the cells mRNA, sample Tags , Abseq barcodes and unique molecular identifiers. cDNA was subjected to several PCR-amplification and clean-up steps using AMPure beads to obtain the three different libraries. In a final OCR step a Illumina i7 sequences was added, library size and concentration were controled  using a Tapestation and Qubit respectively and libraries were pooled together for sequecing.