NextSeq 500 paired end sequencing; Interference of B-Cell and T-Cell Receptor Genes in Single-Cell RNA Sequencing Data
Interference of B-Cell and T-Cell Receptor Genes in Single-Cell RNA Sequencing Data
Venous blood was collected in Lithium-Heparin tubes from the antecubital area of the arm of four patients with untreated early rheumatoid arthritis (RA). PBMCs were isolated using Ficoll (GE Healthcare) and frozen in 10% dimethylsulphoxide (DMSO, Sigma-Aldrich) to be stored at -150° C. Frozen PBMCs were thawed within 50 days of freezing and resuspended in RPMI-1640 10% FCS (Gibco, Thermofisher). Samples were stained with Fixable Viability Dye eFluor™ 506 (Invitrogen) to exclude dead cells.  CD20+CD3- B cells were electronically gated and CD27 versus IgD displayed. In this gate memory B cells were isolated by excluding CD27-IgD+ B cells and sorted as a single population using a Sony H800 cell sorter. Cell suspensions were loaded into a Chromium cell processing unit (10x Genomics, Pleasanton, CA, USA), generating gel bead-in emulsions (GEMs). 5' gene expression libraries were constructed according to standard 10x protocol, using the Chromium Single Cell V(D)J Reagent Kits (v1.1 chemistry, 10x Genomics) GEMs were used to construct sequencing libraries using the standard 10x protocol. Genomics Chromium Single Cell V(D)J Reagent Kits (5', chemistry 1.1).