title: NextSeq 550 paired end sequencing; Single cell sequencing for B cell repertoire of multi-year serial blood samples from two healthy donors.
design: Single cell sequencing for B cell repertoire of multi-year serial blood samples from two healthy donors.
library_construction_protocol: Peripheral blood mononuclear cells (PBMC) from the year 2010 (Donor1) and year 2014 (Donor11) were isolated by the standard density-gradient centrifugation and cryopreserved until the day of use. Blood samples collected in 2020 from both Donor1 and Donor2 were processed immediately to isolate memory B cells and plasma cells by flow cytometry followed by single-cell immune profiling using the 10X system. Single cell suspension was performed according to Chromium Next GEM Single cell V(D)J Reagent kits v1.1. The positive fractions of memory B cells and plasma cells were collected in 6ul of FCS containing eppendorf tubes that were pre-coated overnight with 2percent BSA.  The 5scRNA-seq libraries were generated using Chromium Next GEM Single Cell V(D)J Reagent Kit v1.1 or v2 (10X Genomics) according to the manufacturers protocol. Paired heavy and light chain BCR libraries were prepared from the sorted B cell populations. All the steps were followed as per the manufacturers instructions in user guide recommended for 10X Genomics kit v1.1 or 2.