GSM8160956: SS6, ADT; Homo sapiens; OTHER
PBMCs were isolated blood from five PAPS patients and two HD. Total B cells were isolated with the EasySep™ Human B-cell Isolation Kit (Stemcell Technologies). B cells were resuspended at 10x106/mL and incubated with Human TruStain FcX Fc Blocking reagent for 10min at 4°C. Cells were then incubated with the 10 following antibodies conjugated to an antibody-derived tag for 30 min at 4°C : CD19 (HIB19,TotalSeq, BioLegend) CD27 (0323), CD24 (ML5), CD38 (HIT2), CD5 (UCHT2), HLA-DR (L243), CD86 (IT2.2), IgM (MHM-88), IgD (IA6-2), CD21 (Bu32). The scRNA-seq libraries were generated using Chromium Next GEM Single Cell 5' V(D)J Reagent Kit v.1.1 with Feature Barcoding (10X Genomics) according to the manufacturer's protocol. Gene expression (mRNA), ADT and VDJ BCR libraries were constructed. Briefly, cells were counted and up to 10 000 cells were loaded in the 10X Genomics Chromium Controller to generate single-cell gel-beads in emulsion. After reverse transcription, gel-beads in emulsion were disrupted. Barcoded complementary DNA was isolated and amplified by PCR. Following fragmentation, end repair and A-tailing, sample indexes were added during index PCR. The purified libraries were sequenced on a Novaseq S2 flowcell (Illumina). CITE-Seq + VDJ-Seq