Libraries were prepared as previously described (Mesin et al., 2020 (link)). Briefly, nucleic acids were extracted from sorted single B cells and reverse transcribed into cDNA using RT maxima reverse transcription (Thermo Fisher Scientific) and oligo(dT) primer. Ig heavy chains were amplified by PCR using a consensus forward primer for all V regions and reverse primers specific for each isotype. In the next round of PCR, 5-nucleotide barcodes were introduced using forward and reverse primers to identify the plate number/row position and column position, respectively. In the last round of PCR, Illumina paired-end sequencing adapters were incorporated. PCR products were then pooled, cleaned up using SPRI beads, and sequenced using a 500-cycle Reagent Nano kit v2 on the Illumina Miseq platform according to the manufacturer’s instructions.
Protocol detail
Single B Cell Ig Repertoire Profiling
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B cells
Cdna
Ig heavy chains
Isotype
Nucleic acids
Nucleotide
Oligo
Primers
Reverse transcription
V primer
Corresponding Organization : Rockefeller University
Other organizations : Helmholtz Centre for Infection Research, Massachusetts Institute of Technology, Ragon Institute of MGH, MIT and Harvard, Broad Institute, Boston Children's Hospital, Harvard University, Harvard Stem Cell Institute, Cornell University, Spanish National Cancer Research Centre, Centro de Investigación del Cáncer, Technische Universität Braunschweig, Medizinische Hochschule Hannover
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Variable analysis
independent variables
- Preparation of libraries as previously described (Mesin et al., 2020)
- Extraction of nucleic acids from sorted single B cells
- Reverse transcription of cDNA using RT maxima reverse transcription and oligo(dT) primer
- Amplification of Ig heavy chains by PCR using a consensus forward primer for all V regions and reverse primers specific for each isotype
- Introduction of 5-nucleotide barcodes using forward and reverse primers to identify the plate number/row position and column position, respectively
- Incorporation of Illumina paired-end sequencing adapters in the last round of PCR
- Sequencing of PCR products using a 500-cycle Reagent Nano kit v2 on the Illumina Miseq platform
- Not explicitly mentioned
- No positive or negative controls were explicitly mentioned in the protocol.
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