GSM9009714: scRNA-seq and Cell Hashing, PBMCs, HTO; Homo sapiens; RNA-Seq
Freshly isolated PBMCs were counted, and viability was assessed using trypan blue. Cells were washed and labeled with anti-ꞵ2-microglobulin/anti-CD298 antibodies conjugated to oligonucleotides (TotalSeq-B anti human Hashtag antibodies, Biolegend, Table S3) for 30 min at 4°C according to manufacturer's instructions. Equal numbers of labeled cells from each sample were mixed and diluted in PBS + 0.04% BSA to a final concentration of 1600 cells/µl. The sample was then immediately processed with the Chromium Next GEM Single Cell 3' v3.1 kit, with feature barcoding for cell surface proteins, according to the manufacturer protocol. Single-cell RNA-seq libraries were prepared using the 10X Genomics technology. Final libraries were quantified by qPCR with the NEB-next Library Quant Kit (New England Biolabs), as well as with Qubit and TapeStation. Sequencing was done on a Nova-Seq6000 using SP, 100 cycles kit mode allocating 800M reads in total (Illumina). Fastq files were generated by the usage of bcl2fastq v2.20.0.422.