Illumina MiSeq paired end sequencing; Comparative analysis of B cell receptor repertoires revealed a delay in CD24hiCD38hi transitional B lymphocyte maturation in Multiple Sclerosis patients
Comparative analysis of B cell receptor repertoires revealed a delay in CD24hiCD38hi transitional B lymphocyte maturation in Multiple Sclerosis patients
Venous blood was collected in EDTA Vacutainers (BD). Blood samples were diluted two times in PBS with 2 mM EDTA and layered onto Ficoll-Paque Plus (GE Healthcare) and then centrifuged at 900 g for 40 minutes at room temperature. Isolated PBMCs from MS patients were incubated with ACK lysing buffer for complete deletion of any included red blood cells. Total B-cell pool and regulatory CD19+CD24highCD38high subpopulation were sorted individually for each donor directly into Qiazol lysis reagent (Qiagen, Germany). RNA extraction was performed using RNeasy Mini Kit (Qiagen, Germany) according to manufacturer's protocol Reverse transcription (RT) for cDNA synthesis was run with MMLV RT with oligo(dT) according to the manufacturer's instructions (Evrogen, Russian Federation). Degenerate primers for amplification of the variable fragments of the immunoglobulin repertoire from cDNA were modified from those previously described [Cheng, J. et al. PNAS, 2011]. Fifteen forward VH primers were used separately for each sample with equimolar mixture of four reverse JH primers. Thirteen Vκ primers and sixteen Vλ primers were separately used to amplify the VL genes with corresponding equimolar mixture of two reverse Jκ or three reverse Jλ primers for each sample. Band of ~400 bp (variable fragment of IgH, IgK and IgL) were isolated from an agarose gel. About 1 μg of the PCR product was ligated with adapters from NEBNext Multiplex Oligos using NEBNext Ultra DNA Library Prep Kit for Illumina (NEB).