Sequencing of NP-specific B cells was performed as previously described (Goenka et al., 2014 (link)). In brief, flow cytometry–sorted WT or Itch KO NP+ GC B1-8 cells were lysed and stored in TRIZOL. RNA was isolated using chloroform extraction, precipitation in 70% ethanol, and recovery in RNase-free water. RNA was converted to cDNA using Superscript III reverse transcription (Invitrogen) and pooled constant region-specific primers for the gamma chain to preferentially amplify IgG transcripts (Rohatgi et al., 2008 (link)). cDNA was subjected to two rounds of nested PCR using Expand High Fidelity Polymerase (Roche) with 5′ primers for VH186.2 (Lu et al., 2001 (link)) and 3′ primers for all Ig heavy chain junction region genes (Rohatgi et al., 2008 (link)). Amplified bands of ∼350 bp were purified using Qiaquick gel extraction kit (Qiagen) and cloned into pCR-TOPO vector using the TOPO TA Cloning kit (Invitrogen). Plasmid was prepared from individual bacterial colonies using a QiaPrep spin miniprep kit (Qiagen), and Ig heavy chain variable gene inserts were sequenced at the CHOP Nucleic Acid and Protein Core Facility. Mutations were analyzed using the International Immunogenetics Information System V-Quest database (Brochet et al., 2008 (link)).