title: non-progressor GEM8 myeloid cells scRNAseq
design: PBMCs samples stored in liquid nitrogen at -140C were thawed and resuspended in fluorescent activated cell sorting (FACS) buffer. Myeloid cells were isolated by FACS sorting on Aria sorter by forward scatter and side scatter, based on their larger cell size compared to lymphocytes. Myeloid cells were counted manually utilizing a hemocytometer and a volume corresponding to 60,000 cells was loaded in the Chromium Next GEM Chip G Single Cell Kit (10X Genomics, Cat# 1000127). The chip was loaded on a Chromium controller where individual cells are isolated into droplets together with gel beads coated with unique primers bearing 10X cell barcodes, UMI (unique molecular identifiers) and poly(dT) sequences. Reverse transcription reactions allow then to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up. Bulk cDNA is amplified and cleaned up. Indexed sequencing libraries will be constructed using the reagents from the Chromium Next GEM Single Cell 3 Reagent Kits v3.1 (Dual index; 10X Genomics, Cat# 1000268). Indexed libraries were equimolarly pooled and sequenced on an Illumina Novaseq S4 using paired-end 28x90bp as sequencing mode aiming at  20,000 paired reads per cell and ~20,000 cells per each library.
identifiers.namespace: SUB15101377
identifiers.id: GEM8