GSM6837113: PBMCspleen, HTO-derived cDNA; Homo sapiens; OTHER
Cells were isolated form PBMCs and splenocytes of four patients (two healthy donors, HD1 18 years old male and HD2 61 years old female; two brain-dead donors, BD1 57 years old female, and BD2 58 years old female). CITE-seq using Hashtag Oligos (HTO) was then performed to pool samples into a single 10X Genomics channel for scRNAseq as described in Abidi et al. (Abidi et al. 2020). HTO binding was carried out according to the manufacturer's guidance (BioLegend, Total-seq B) Basically, after thawing ,and cell washing, 1M cells were centrifuged and resuspended in 100uL PSE buffer (PBS/FBS/EDTA). Cells were incubated with 10ul human FcR blocking reagent for 10 min at 4°C. 1 uL of a Hashtag oligonucleotide (HTO) antibody (Biolegend) was then added to each sample and incubated at 4°C for 30 min. Cells were then washed in 1mL PSE, centrifuged at 500g for 5 min and resuspended in 200uL PSE . Cells were stained with 2uL DAPI, 60uM filtered and viable cells were sorted (ARIA). Cells were checked for counting and viability then pooled and counted again. Cell viability was < 95%. Cells of HD1 Spleen were lost during the washing step and thus not used during further downstream processing. Cells were then loaded on 1 channel of Chromium Next Controller with 3' single cell Next v3 kit (10x Genomics Inc. Pleasanton, CA). We then followed the supplier's protocol CG000185 Rev C until library generation. For HTO library, we followed the protocol of 10x genomics 3' feature barcode kit (PN-1000079) to generate HTO libraries. 310pM of pooled libraries were sequenced on a NOVAseq6000 instrument with a S1 (v1) flow-cell (Illumina Inc. San Diego, CA) at the genomic platform core facility on the basis of a placement agreement (Nantes, IRS-Un, France) . The program was runned as follows : Read1 29 cycles / 8 cycles (i7) /0 (i5)/Read2 93 cycles (Standard module, paired-end, 2 lanes).