For PBMCs and CD19+ B cells: PBMCs were isolated from 110 ml of whole blood by centrifugation over Ficoll. CD19+ B cells were isolated by positive selection using magnetic beads as previously described36 (link). Total RNA was extracted from each sample using an RNeasy mini kit (Qiagen) with quality assessed using an Agilent BioAnalyser 2100 and RNA quantification performed using a NanoDrop ND-1000 spectrophotometer.
For flow-sorted B cell samples from Espéli et al.37 (link): Flow sorting was performed using CD19-BV785, CD38-BV711, CD3-NC650, CD14-605NC, CD24-PerCP-Cy5.5, IgD-FITC. CD27-PE-Cy7 and Aqua (Invitrogen) (flow protocol outlined inExtended Data Figure 1 ), into sorting buffer (10mM Tris pH 8.0 and RiboLock RNase Inhibitor (1U/μL)) and frozen immediately.
Total IgA and IgE levels in patient serum were measured using a ProcartaPlex immunoassay kit (ThermoFisher) using 25ul of serum from each individual and run on a Luminex xMAP analyser. Raw data (MFI) were normalised to a concurrently measured 7 point standard curve according to the manufacturer’s instructions to return an absolute quantification (pg/ml). All measured values were encompassed by the standard distribution.
For flow-sorted B cell samples from Espéli et al.37 (link): Flow sorting was performed using CD19-BV785, CD38-BV711, CD3-NC650, CD14-605NC, CD24-PerCP-Cy5.5, IgD-FITC. CD27-PE-Cy7 and Aqua (Invitrogen) (flow protocol outlined in
Total IgA and IgE levels in patient serum were measured using a ProcartaPlex immunoassay kit (ThermoFisher) using 25ul of serum from each individual and run on a Luminex xMAP analyser. Raw data (MFI) were normalised to a concurrently measured 7 point standard curve according to the manufacturer’s instructions to return an absolute quantification (pg/ml). All measured values were encompassed by the standard distribution.