Nearly 1500-10,000 sorted HA-specific mature B cells from individual organs were processed into single cells in a chromium controller (10X genomics). During this process, individual cells are embedded in Gel Beads-in-emulsion (GEMs) where all generated cDNA share a common 10X oligonucleotide barcode. After amplification of the cDNA, 5′gene expression library and enriched B cell library, with paired heavy and light chain were generated from cDNA of the same cell using Chromium single cell VDJ reagent kit (V1.1 chemistry, 10X genomics). The 5′gene expression libraries were sequenced in NextSeq or NovaSeq6000 sequencer (Illumina) using NextSeq 500/550 v2.5 sequencing reagent kit (2 × 75 bp) or NovaSeq S1 sequencing reagent kit (2 × 100 bp) (Illumina) respectively. The enriched B cell libraries were sequenced in NextSeq or MiSeq sequencer using NextSeq Mid Output v2.5 sequencing reagent kit (2 × 150 bp) or MiSeq Reagent Kit v2 (2 × 150 bp) (Illumina) respectively.
Lungs from mice M0_1, M7_2 and M14_1 and spleen from M28_3 failed to yield good quality GEMs and libraries and were not sequenced.
Lungs from mice M0_1, M7_2 and M14_1 and spleen from M28_3 failed to yield good quality GEMs and libraries and were not sequenced.