Maxima rt reverse transcriptase

Single GC B cells were index-sorted into 96-well plates containing 5 ml TCL buffer (QIAGEN) supplemented with 1% β-mercaptoethanol. SPRI beads were used to extract nucleic acids as previously described^{25 (link),49 (link)}. RT maxima reverse transcriptase (Thermo Scientific) with oligo(dT) as a primer was used for reverse-transcription into cDNA. A forward primer mix comprising of consensus sequences for all V-regions and reverse primers for each isotype was used to PCR amplify Igh genes. To confirm clonality or for antibody production, Igk genes were amplified separately where needed. Afterwards, 5-nucleotide barcodes were introduced by PCR to label Ig-sequences with plate- and well-specific barcodes. The forward and reverse primers contained barcodes to identify the plate and row number, and barcodes to identify the column position, respectively (adapted from Han et al., 2014). A final PCR step was performed to incorporate Illumina paired-end sequencing adapters into single-well amplicons. PCR-products from all plates were pooled together and subsequently cleaned-up using a 0.7x volume ratio of SPRI beads. Sequencing was performed on the Illumina Miseq platform with a 500-cycle Reagent Nano kit v2 as per the manufacturer’s instructions. Primer sequences are provided separately.