109 B-cell repopulation after rituximab in rheumatoid arthritis Supplementary data Figure S1 | A) Schematic representation of the high-throughput UMI-based BCR repertoire sequencing workflow. Specific reverse transcription of BCRh RNA molecules is performed with UMI-tagged primers complementary to the BCR heavy chain joining gene (step 1). Exonuclease I treatment is performed to remove unbound reverse transcription primers (step 2) before performing a multiplexed PCR with 6 BCRh variable (IGHV) chain forward primers and a single reverse primer (step 3). Obtained amplicons are then indexed with i7 and i5 Nextera Illumina indexes (step 4) and sequenced on an Illumina MiSeq platform. B-C) Scatter plots depicting the clonal overlap in the same pre-treatment (B) or post-treatment (C) sample amplified with or without addition of carrier RNA from the non BCR-expressing cell line HEK939T prior cDNA synthesis. X- and Y-axes depict the frequency of each BCR clonotype in the repertoire as percentage of total UMIs. 5
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