Illumina NovaSeq 6000 paired end sequencing; Longitudinal scRNAseq profiling of memory B cells in severe COVID19 patients
Longitudinal scRNAseq profiling of memory B cells in severe COVID19 patients
Peripheral (CD3-CD14-CD15-CD56-CD19+IgD-) B cells were FACS-sorted (MA900, Sony) in PBS/0.08% FCS from 4 patients (S-CoV) at baseline (M0) and 6 months (M6). 5x104 to 10x105 cells were obtained for each subset. Cells were counted and up to 20 000 cells were loaded in the 10x 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. The scRNA-seq libraries were generated using Chromium Next GEM Single Cell V(D)J Reagent Kit v.1.1 with Feature Barcoding (10x Genomics) according to the manufacturerâ€™s protocol. Gene expression (mRNA), ADT and VDJ BCR libraries were constructed.