PV16_IgG4_JRStanLab
Primer sequences are in Supplemental Table 5. Total RNA was isolated from peripheral blood mononuclear cells collected from 50 ml of blood using RNeasy Midi Kit (Qiagen) as previously described (protocol 9.2 ref (23)). cDNA was synthesized from 1-5 µg of RNA using a SuperScript RT II kit with an oligo-dT primer (Invitrogen). For PV1, PV3, PF1 and PF4, pan-IgG VH repertoires were PCR-amplified using primer sets slightly modified from those previously described (23). IgG1-VH and IgG4-VH repertoires were amplified separately for PV8 and PV16 using isotype specific primer sets for nested PCR.  In some cases APD panned libraries were subjected to NGS. The VH coding sequences (sometimes also including the VL sequences) in pCOMB3x phagemids from PV3a and PV8 libraries were amplified by PCR. PCR reactions were carried out using Q5 DNA Polymerase (New England Biolabs) and products were purified using QIAquick PCR purification kit (Qiagen).     Two NGS platforms were used in this study. Pan IgG VH repertoires (PV1a, PV3, PV3a, PF1, PF1a, PF4, PF4a, PF4b), IgG1 or IgG4 VH repertoires (PV8, PV16), and the VH regions of PV8 panned phage libraries were sequenced using Illumina Miseq 300bp paired end sequencing (Institute for Genome Science, University of Maryland; Next-Generation Sequencing Core, University of Pennsylvania). The VH regions of PV3a panned phage libraries were sequenced using Pacific Biosciences (Pacbio) sequencing (Yale Center for Genome Analysis).