Mus musculus: Swiss webster, Igh Germ Free day42-43: Antibody Heavy Chain Sequences from Lamina propria B cells
LP B cells: DAPI-CD19+B220+IgM+CD93-CD95-GL7-CD23+ were sorted from germ-free (GF) Swiss Webster (SW) at the age of d42-d43. Total RNA was extracted from sorted B cells using TRIzol reagents. The RNA was reverse transcribed by Superscript III reverse transcriptase with Oligo dT primer. The first-strand cDNA was purified by the Agencourt RNAClean XP beads to remove the primers. 19 forward degenerate primers binding to the framework region 1 of the VH region barcoded by a unique 8 nucleotide molecular identifier (UMI) and connected with the universal primer were used for the second strand cDNA synthesis and barcoding. After removing the residual primers by RNAClean XP, the barcoded cDNA was amplified for 25 cycles by constant region primer and universal primer to add 5  and 3  adaptors. The PCR products were purified by QIAquick PCR purification kit, and amplified again for 18 cycles to add Illumina linkers (P5 and P7) and sample barcodes. 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 based on 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 v3 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 250nt from each end.