Hiseq 2500 high output platform

Freshly sorted 1M or 4M P14s were (as indicated above) were resuspended in TRIzol and RNA was purified using an RNAeasy kit (QIAGEN) according to the manufacturer’s protocol. RNA was assessed for purity and quality using an Agilent 2100 Bioanalyzer and RNA seq was performed using the single cell/low input library kit (NEB), full-length cDNAs were sequenced on the Illumina HiSeq 2500 High-output platform using 2 × 150 paired-end libraries. The sequence reads quality was checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and were aligned to the mouse genome version mm10 using STAR aligner (Dobin et al., 2013 (link)). Following read alignment, gene expression profiles were computed using featureCounts (Liao et al., 2014 (link)). Filtering and visualization of differentially expressed genes with a log2fold change >1.5 < −1.5 and a p value < 0.05 were identified using Partek GS software and for some figures values from Partek were normalized and displayed using GraphPad Prism. Gene set enrichment and functional assignment were performed in DAVID bioinformatics resources and software from the Broad Institute as described (Martin and Badovinac, 2016 (link); Shan et al., 2017 (link); Subramanian et al., 2005 (link)). Tissue resident memory gene set (Table 2) was developed from review of existing literature (Urban et al., 2020 (link)).

Sorted live CD45⁺B220⁺ cell populations, pooled from three mice, were loaded onto a 10X Genomics Chromium Controller, and the VDJ library was prepared according to the manufacturer’s guidelines. Samples were sequenced using the Illumina HiSeq 2500 High Output platform. Transcript alignment and generation of feature–barcode matrices were performed using the 10X Genomics CellRanger workflow.
The J1KK monoclonal antibody was cloned from the dominant BCR sequence as either mouse IgA or IgG1 into a pRV-IgK-T2A-IgH-IRES-GFP plasmid (Genscript) and transduced into HEK293T cells. IgA and IgG1 antibodies were purified from serum-free supernatant using a Protein L spin column (Thermo Fisher) and Protein A Plus spin column (Thermo Fisher), respectively, according to the manufacturer’s instructions.

Sorted cell populations were loaded onto a 10x Genomics Chromium Controller, and 5′ gene expression, VDJ, and ADT (for samples in Fig. 4) were prepared according to the manufacturer’s guidelines. Samples used in Fig. 3 were sequenced using an Illumina NextSeq 500 platform. Samples used in Fig. 4 were sequenced using an Illumina HiSeq 2500 High Output platform. The 10x Genomics Cell Ranger workflow was then used for transcript alignment and the generation of sparse matrices for downstream analysis.