title: MK06_U_AAYFK
design: Peripheral blood was obtained from HD and MG subjects. B cells subsets were isolated using flow cytometry. Briefly, peripheral B cells from MG patients and healthy donors were first enriched to >95% by positive selection using CD20 magnetic beads (Milteny). The cells were then stained with perCP-Cy5.5 antihuman CD27 (clone O323), Pacific Blue anti-human CD19 (clone HIB19) (both from Biolegend). Nave (CD19+, CD27-) or memory B cells (CD19+, CD27+) were then sorted on a FACSAria flow cytometer (BD) into PCR tubes and immediately frozen on dry ice. RNA (250 ng) was reverse-transcribed into cDNA using a biotinylated oligo dT primer. An adaptor sequence was added to the 3' end of all cDNA, which contains a universal priming site and a 17-nucleotide unique molecular identifier (UMI). Products were purified using streptavidin-coated magnetic beads followed by a primary PCR reaction using a pool of primers targeting the IGHA, IGHD, IGHE, IGHG, IGHM, IGKC and IGLC regions, as well as a primer against the universal adaptor sequence. The immunoglobulin-specific primers contained tail sequences for later Illumina adaptor addition. PCR products were then purified using AMPure XP beads. A secondary PCR was then performed to add the Illumina P5 adaptor to the constant region end and a sample-indexed P7 adaptor to the universal adaptor end. The number of secondary PCR cycles was tailored to each sample to avoid entering plateau phase, as judged by a prior quantitative PCR analysis. Final products were purified, quantified with a TapeStation (Agilent Genomics) and pooled in equimolar proportions, followed by high-throughput 2x300 base-pair paired-end sequencing with a 20% PhiX spike on the Illumina MiSeq platform according to manufacturers recommendations, except for performing 325 cycles for read 1 and 275 cycles for read 2.