GSM8733940: Multiplexed_PBMCs(HC_S_NS); Homo sapiens; RNA-Seq
Cell stocks were thawed in 37℃ 10% FBS/DMEM. Samples were washed twice with cold Ca2+ and Mg2+ free 0.04 % BSA/PBS at 300g for 5min at 4°C. Samples were gently resuspended in cold Ca2+ and Mg2+ free 0.04% BSA/PBS, and then counted using a LUNA-FX7™ Automated Fluorescence Cell Counter (Logos Biosystems) with AO/PI staining. Multiplexing individual samples for scRNA-seq To multiplex samples, each sample was tagged with antibody-polyadenylated DNA barcode for human cells (BD Biosciences, cat no. 633781). Briefly, cells were stained with multiplexing antibody for 20 min at room temperature, and then washed 3 times by using stain buffer (BD Biosciences, cat no. 554656). After final washing, samples were gently resuspended in cold sample buffer (BD Biosciences, cat no. 664887), counted with a LUNA-FX7™ Automated Fluorescence Cell Counter (logos biosystems), and then pooled. Single cell capture and cDNA synthesis Single cell capture was performed using BD Rhapsody Express instrument according to the manufacturer's instruction (BD Bioscience). Briefly, pooled cells from each sample in cold sample buffer were loaded into the BD Rhapsody cartridge (BD Biosciences, cat no. 664887). After cells separation, cell barcode-magnetic beads were loaded into the cartridge. Cells were lysed and the mRNA capture bead were retrieved. cDNA synthesis and Exonuclease I treatment were performed on the mRNA capture beads using BD Rhapsody cDNA kit (BD Biosciences, cat no. 633773). The purified WTA and Sample tag libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA), and qualified using the Agilent Technologies 4200 TapeStation (Agilent technologies). The libraries were sequenced using NovaSeq platform (Illumina).