High sensitivity d1000 screentape assay

Total DNA was isolated from each sample using the PowerSoil^® DNA Isolation Kit (Catalogue nº 12888-50; Mo Bio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol. Libraries preparation was performed using the amplification of the V4 regions of the 16S rRNA gene with the primers set 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGG TWTCTAAT-3′) following the method described in [37 (link)]. The size, purity, and concentration of the libraries were assessed using the D1000 High Sensitivity ScreenTape Assay (Agilent, Santa Clara, CA, USA). Normalized libraries were pooled and amplicons (end pairs: 300 × 300 bp) were sequenced in the Illumina MiSeq platform by MR DNA (www.mrdnalab.com, Shallowater, TX, USA). All sequencing data were submitted to the NCBI SRA database (accession number PRJNA875592).

Naive splenic B cells were isolated from the PCT64H/L knock-in mouse line using the mouse, immunomagnetic negative selection, Pan-B cell Isolation Kit (Miltenyi Biotec, San Diego, CA). The B cell suspension was diluted 1:10 and cell density was manually determined using a hemocytometer. Cell viability was determined to be >90% by automated cell counter (Nucleocounter, Chemometec). Approximately 10,000 were loaded into the 10x Genomics Chromium Controller and encapsulated in gel beads in emulsion. Single-cell gene expression libraries were prepared using the Chromium Single-cell 5′ Library and Gel Bead Kit following the manufacturer’s user guide (10x Genomics, Pleasanton, CA). The integrity of the library was determined using the D1000 high sensitivity ScreenTape assay (Agilent, Santa Clara, CA) and quantified using the Qubit fluorometry assay (AAT Bioquest, Sunnyvale, CA). BCR libraries were sequenced on the MiSeq System (Illumina, San Diego, CA) with 2 × 150 paired end reads (Genewiz, South Plainfield, NJ). Sequencing data produced from the Chromium Single Cell 5′ V(D)J library was analyzed using a customized the 10x Genomics Cellranger pipeline that includes the LMCA PCT64 heavy chain sequence (Genewiz, South Plainfield, NJ).

On postnatal day 1 offspring were euthanized, the entire gastrointestinal tract was excised, and the ileum was rapidly frozen in liquid nitrogen and maintained in –80 °C until RNA isolation. For bulk RNA sequencing analysis, tissues were homogenized in Trizol, and messenger RNA was purified using the RNeasy Mini kit (Qiagen). Illumina single-end cDNA libraries of postnatal day 1 ileum mRNA was prepared from 250 ng total RNA using the TruSeq Stranded mRNA Kit with poly-A enrichment (RS-122-2101, Illumina) according to the manufacturer’s protocol. Library fragment size was quantified using an Agilent High Sensitivity D1000 ScreenTape Assay on an Agilent 4200 Tapestation System (G2991AA). Library concentration was quantified using the Qubit dsDNA HS (High Sensitivity) Assay Kit. Samples representative of all treatment groups were multiplexed and sequenced on an Illumina NextSeq500 instrument using high-output 1 × 75 bp geometry (Illumina).