Nextera indexing kit

Shotgun metagenomic library was constructed from fecal DNA with the Nextera DNA sample preparation kit (Illumina, San Diego, CA), as per manufacturer’s specification. Barcoding indices were inserted using Nextera indexing kit (Illumina). Products were purified using Agencourt AMpure XP kit (Beckman Coulter, Brea, CA) and pooled for sequencing. Samples were sequenced using MiSeq reagent kit V2 (Illumina) in a HiSeq2500 sequencer.
Raw sequences were sorted using assigned barcodes and cleaned up before analysis (barcodes removed and sequences above a quality score, Q ≥ 30 taken forward for analyses). For assembly and annotation of sequences, MetAMOS^{47 (link)} pipeline or Partek Flow software (Partek® Flow®, Partek Inc., St. Louis, MO) were used. These softwares provide powerful tools to filter unique hits between human and mouse-specific genes versus microbial signatures. Alpha and Beta diversity calculations were done using embedded programs within the metagenomic pipeline, or using Stata15 (StataCorp LLC, College Station, TX) or EXPLICET software.^{48 (link)} Functional profiling was performed using HUMAnN2-0.11.1^{49 (link)} with Uniref50 database to implement KEGG orthologies.

Single cell suspensions from the spleens of immunized mice were stained with (NANP)_n tetramers and antibodies to B cell markers as described in the supplementary experimental procedures. Antigen specific cells were sorted on a FACS ARIA I or II instrument prior to RNA extraction with the Arturus Picopure RNA isolation kit (Invitrogen) and cDNA preparation using the iScript cDNA synthesis kit (BioRad). BCR sequences were amplified using previously described heavy and kappa chain primers including adaptor sequences allowing subsequent indexing using the Nextera indexing kit (Illumina). Analysis was performed in house using R-scripts and the program MiXCR as described in supplementary experimental procedures.

DNA isolation, library preparation, and sequencing were carried out at CosmosID (Germantown, MD) using a vendor-optimized protocol. Briefly, plaque samples were spiked with Truepera radiovictrix, Imtechella halotolerans, and Allobacillus halotolerans using ZymoBIOMICS Spike-in Control II (Zymo Research, Irvine, CA) to enable bacterial cell number quantification. To enhance bacterial cell lysis, plaque samples were incubated with MetaPolyzyme at 35 °C for 12 h, and DNA was extracted employing the ZymoBIOMICS DNA MicroPrep with bead-beating according to the manufacturer’s instructions. The concentration of all DNA samples and libraries were determined using the Qubit dsDNA HS assay and Qubit 4 fluorometer (ThermoFisher Scientific, Waltham, MA). To construct DNA libraries, 1 ng of input genomic DNA was fragmented, amplified, and indexed utilizing Nextera XT DNA Library Preparation and Nextera Indexing Kit (Illumina, San Diego, CA). DNA libraries were purified using AMPure magnetic beads (Beckman Coulter, Brea, CA) and then normalized for equimolar pooling. Sequencing was performed with a HiSeq sequencer (Illumina), targeting a coverage of 3 − 4 million paired-end 2 × 150 bp reads.

Exon-specific primers linked to Illumina sequencing adapters were designed to generate amplicons spanning potentially novel exons using cDNA from day 0 (see Additional file 6). Primers were used in both convergent and divergent orientation to enable amplification of both circular and linear isoforms. Illumina sequencing indexes were added to each amplicon using a Nextera indexing kit (Illumina), and amplicons were sequenced to a target depth of 1500-6000 reads (depending on the number and intensity of visible products) using a MiSeq Nano Kit (Illumina) all according to manufacturer’s recommendations.

Shotgun metagenomic library was constructed from fecal DNA with the Nextera DNA sample preparation kit (Illumina, San Diego, CA), as per manufacturer’s specification. Barcoding indices were inserted using Nextera indexing kit (Illumina). Products were purified using Agencourt AMpure XP kit (Beckman Coulter, Brea, CA) and pooled for sequencing. Samples were sequenced using MiSeq reagent kit V2 (Illumina) in a HiSeq2500 sequencer.
Raw sequences were sorted using assigned barcodes and cleaned up before analysis (barcodes removed and sequences above a quality score, Q ≥ 30 taken forward for analyses). For assembly and annotation of sequences, MetAMOS [28 (link)] pipeline or Partek Flow software (Partek^® Flow^®, Partek Inc., St. Louis, MO) were used. These softwares provide powerful tools to filter unique hits between human and mouse-specific genes versus microbial signatures. Alpha and Beta diversity calculations were done using embedded programs within the metagenomic pipeline, or using Stata15 (StataCorp LLC, College Station, TX) or EXPLICET software [29 (link)].
Functional profiling was performed using HUMAnN2-0.11.1 [30 (link)] with Uniref50 database to implement KEGG orthologies.