Mus musculus: Mixed C57BL/6 x 129 genetic background , Igh wt VJκ5:Antibody Heavy Chain Sequences from Bone marrow Pro-B cells
Pro B lymphocytes (DAPi-B220+CD19+BCR-CD43+) and follicular B lymphocytes (DAPi-CD19+BCR+CD93-CD23hiCD21int) sorted from Igh wt VJk5 mice were sequenced using the Illumina MiSeq platform. RNA was reverse-transcribed into cDNA using immunoglobulin heavy 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 chains were barcoded and residual primers removed by purifying with RNAClean XP beads. Qiaquick PCR purification kit was used to purify the pre-amplification products. Subsequently, first round PCR added 5’ and 3’ adaptors to both ends of the amplicon and resulted in amplification of heavy chains using 25 cycles. Second round PCR added Illumina linkers (P5 and P7) and sample barcodes to heavy chains amplified using 17 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 base calling. The libraries were sequenced from both ends of the molecules to a total read length of 255nt from each end.