title: Illumina HiSeq 2500 paired end sequencing; Longitudinal full-length IGH repertoire profiling and clonal lineage dynamics in memory B cells, plasmablasts and plasma cells of human peripheral blood
design: Longitudinal full-length IGH repertoire profiling and clonal lineage dynamics in memory B cells, plasmablasts and plasma cells of human peripheral blood
library_construction_protocol: Venous blood was collected in EDTA Vacutainers (BD). Blood samples were diluted two times in PBS with 2 mM EDTA and layered onto Ficoll (Paneco) and then centrifuged at 300 g for 30 minutes at room temperature. Cell were separated with fluorescence activated cell sorting (BD FacsAria III, BD Biosciences) into the following subpopulations: memory B-cells (Bmem, CD19+ CD20+ CD27+ CD138-), plasmablasts (PBL, CD20 CD19+ CD27++ CD138), plasma cells (PL, CD20 CD19 Low/ CD27+ CD138+) individually for each donor directly into RLT buffer (Qiagen, Germany). RNA extraction was performed using RNeasy Mini Kit (Qiagen, Germany) according to manufacturers protocol Immunoglobulin heavy chain (IGH) cDNA libraries were prepared according to the protocol described earlier (Turchaninova et al. 2016) with several modifications. Briefly, we used 5'-RACE (Rapid Amplification of cDNA Ends) approach based on a template-switch effect to introduce 5 adaptors during cDNA synthesis. These adaptors contain both unique molecular identifiers (UMIs), allowing error-correction, and sample barcodes (see Zvyagin et al. 2017 for the adaptor structure) for ruling out cross-sample contaminations. Universal sequence for annealing of forward PCR primer was also introduced by the adaptor. The set of primers specific for C-segment was used in an multiplex RT reaction followed by PCR amplification step with another semi-nested set of C-segment specific primers preserving isotype information.