title: NextSeq 550 paired end sequencing; Single cell sequencing for B cell repertoire of multi-year serial blood samples from a healthy donors.
design: Single cell sequencing for B cell repertoire of multi-year serial blood samples from a healthy donors.
library_construction_protocol: Peripheral blood mononuclear cells (PBMC) from june 2018, november 2018 and februaru 2021 were isolated by the standard density-gradient centrifugation and cryopreserved until the day of use. Blood samples collected to isolate plasmablasts (and memory B cells for february 2021 sample) 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 2% BSA. The 5'scRNA-seq libraries were generated using Chromium Next GEM Single Cell V(D)J Reagent Kit v1.1 or v2 (10X Genomics) according to the manufacturer's protocol. Paired heavy and light chain BCR libraries were prepared from the sorted B cell populations. All the steps were followed as per the manufacturer's instructions in user guide recommended for 10X Genomics kit v1.1 or 2.