Mus musculus 17/9 DO11.10 RAG-/- ova immunized ova negative sorted HA-specific monoclonal splenic B cell immunoglobulin heavy chain variable region
Ova negative B lymphocytes isolated from immunized 17/9 DO11.10 RAG-/- mice were sequenced using the Illumina MiSeq platform. RNA was reverse-transcribed into cDNA using immunoglobulin heavy and light chain constant region specific primers.  Reverse transcriptase was heat inactivated and residual primers removed using Uracyl DNA glycosylase.  The glycosylase was then heat inactivated. Immunoglobulin heavy and light chains were barcoded separately and residual primers removed using Exonuclease VII.  Subsequently, first round PCR added 5Í and 3Í adaptors to both ends of the amplicon and resulted in amplification of heavy and light chains in separate reactions using 18 cycles. Second round PCR added Illumina linkers (P5 and P7) and sample barcodes to heavy and light chains amplified in separate reactions using 19 cycles. Products were quantified on a Qubit fluorimeter, run on a fragment analyzer with amplicon regions and expected sizes confirmed. Samples were then pooled in equal amounts according to product concentration. The pooled products were then size selected on an agarose gel and purified by gel extraction. Purified, size-selected products were run on an Agilent Bioanalyzer to confirm appropriate profile and determination of average size. The final pools were quantitated using Qubit and diluted to 5nM final concentration. The 5nM dilutions were further quantitated by qPCR on a BioRad CFX Connect Real-Time System and pooled evenly. The pool was denatured and spiked with 15% non-indexed PhiX control library provided by Illumina and loaded onto the MiSeq V2 Nano flowcell at a concentration of 7 pM for cluster formation and sequencing. The PhiX control library provides a balanced genome for calculation of matrix, phasing and prephasing, which are essential for accurate basecalling. The libraries were sequenced from both ends of the molecules to a total read length of 250nt from each end.