Novaseq 6000 s2 flow cell

PCR amplification of the 16S rRNA gene V3-V4 hypervariable region was performed using dual-barcoded universal primers 318F and 806R as previously described (84 (link)). In brief, amplicon pools were prepared for sequencing with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA), and the size and quantity of the amplicon library were assessed on the LabChip GX system (PerkinElmer, Waltham, MA) and with a library quantification kit for Illumina (Kapa Biosciences, Woburn, MA), respectively. The PhiX control library (v3) (Illumina, San Diego, CA) was combined with the amplicon library. High-throughput sequencing of the amplicons was performed on an Illumina MiSeq platform using the 300-bp paired-end protocol. Sequence libraries were prepared from the extracted DNA using the Nextera DNA Flex kit (Illumina, San Diego, CA) according to the manufacturer’s specifications. Libraries were then pooled in equimolar proportions and sequenced on a single Illumina NovaSeq 6000 S2 flow cell providing an average of 6.5 million pairs of 150-bp reads per library at the Genomic Resource Center at the University of Maryland School of Medicine.

Genomic DNA was fragmented to a size range of 150–250 bp using the Covaris S220 instrument. The fragmented DNA was end repaired and adenylated. The SureSelect Adaptor was then ligated followed by a pre-amplification. After library preparation, target regions were captured by hybridization of biotinylated baits (library probes) using Agilent SureSelect XT Human All Exon V6 (Agilent, Santa Clara, CA, USA). Captured target sequences were then isolated using streptavidin-coated magnetic beads. Subsequently, the appropriate 8-bp single index tags were added during sequencing library amplification. All steps were done using Agilent SureSelect XT Reagent Kit (Agilent, Santa Clara, CA, USA). Final quality control was done using Qubit 3 fluorometer and Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The libraries were sequenced in paired-end mode (2 × 50 nt) on a NovaSeq6000 S2 Flowcell (Illumina, San Diego, CA, USA) resulting in ~200 million distinct sequencing reads per library.

The jejunum and colon tissues of the mice were dissected according to their gastrointestinal anatomical features. Intraluminal stool contents were collected from dissected tissues and stored immediately in DNA/RNA Shields (Zymo Research, Irvine, CA, USA) at −80°C to stabilize and protect the integrity of nucleic acids and minimize the need for immediate processing or freezing of specimens. DNA extraction was described previously [30 (link), 62 (link)]. In brief, 0.15–0.25 grams of fecal samples were extracted using the Quick-DNA Fecal/Soil Microbe kit (Zymo Research, Irvine, CA, USA). Negative extraction controls were included to ensure that no exogenous DNA contaminated the samples. Metagenomic sequencing libraries were constructed using the Nextera XT Flex Kit (Illumina), according to the manufacturer’s recommendations. Libraries were then pooled together in equimolar proportions and sequenced on a single Illumina NovaSeq 6000 S2 flow cell at Maryland Genomics at the University of Maryland School of Medicine.

Up to 75 ng of sheared genomic DNA was subjected to bisulfite conversion using the EZ-96 DNA Methylation Kit (Zymo Research), with liquid handling on a MicroLab STAR (Hamilton). Dual-indexed sequencing libraries were prepared using Accel-NGS Methyl-Seq DNA library preparation kits (Swift BioSciences) and custom liquid handling scripts executed on the Hamilton MicroLab STAR. Libraries were quantified using KAPA Library Quantification Kits for Illumina Platforms (Kapa Biosystems). Four uniquely dual-indexed libraries, along with the 10% PhiX v.3 library (Illumina), were pooled and clustered on an Illumina NovaSeq 6000 S2 flow cell followed by 150 bp, paired-end sequencing. Total read count and average sequencing depth (in read pairs), as well as percentage of CpGs, per sample, at 1× and 10×, are detailed in Supplementary Table 1. Also listed are average methylation levels, per sample, at CpG, nonCpG and CC dinucleotides. Intriguingly, sorted neuron samples showed higher CpA methylation (approximately 10%) compared with other samples (approximately 1%).