Ampure xp magnetic beads

Total DNA was extracted from jejunal contents (200 mg/sample) using a QIAamp® Fast DNA Stool Mini kit (Qiagen Inc.). The DNA concentration was measured by a NanoDrop spectrometer (Thermo Scientific™, USA). The 16S amplicon PCR (targeting the V3-4 region) was performed as per the Illumina 16S metagenomic sequencing library preparation protocol. The PCR products were purified using AMPure XP magnetic beads (Beckman Coulter, Inc, CA, USA). The size of the amplicons was confirmed by gel electrophoresis. Sequence libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, USA) with 24 or 96 indexes as per the manufacturer’s instructions. The libraries were cleaned up with AMPure XP magnetic beads and analyzed by a bioanalyzer using a high sensitivity DNA kit (Agilent, Santa Clara, CA). The concentration of the amplicon sequence libraries was measured using the Qubit dsDNA BR (broad range) assay kit (Thermo Fisher Scientific, Waltham, USA). The normalized libraries were pooled, denatured with 0.2N NaOH, diluted using H1 hybridization buffer and loaded onto the MiSeq V3 (600 cycles) cartridge. Sequencing was performed using the Illumina MiSeq platform (Illumina, San Diego, US) as per the company’s protocol. The sequence quality was examined in real time using the Sequencing Analysis Viewer (SAV) V1.8.

Amplicons from MS‐RT‐PCRs were cleaned by 0.45× of Agencourt AMPure XP Magnetic Beads (Beckman Coulter) according to manufacturer's protocol and eluted in 30 µl of HyClone molecular biology water (Genesee Scientific). Concentration of the eluate was measured using the Qubit dsDNA HS Assay kit (ThermoFisher) on the Qubit 3.0 fluorometer (ThermoFisher). Amplicons were normalized to 0.2 ng/µl. Adapters were added by tagmentation using Nextera XT DNA library preparation kit (Illumina). The reaction was set up using 40% of the suggested final volume. Libraries were purified using 0.7× Agencourt AMPure XP Magnetic Beads, and fragment size distribution was analyzed on the Agilent Bioanalyzer using the High Sensitivity DNA kit (Agilent). Next, samples were normalized to 4 nM and pooled. The loading concentration of the pooled libraries was 15 pM. Libraries were sequenced using the MiSeq v2, 300 cycle reagent Kit (Illumina) in a paired‐end fashion (150 × 2).

RNA sequencing was performed at the Johns Hopkins University School of Medicine Transcriptomics and Deep Sequencing Core, guided by the direction of Dr. Haiping Hao. RNA‐seq library for Illumina platform sequencing was prepared using Illumina TruSeq stranded total RNA Sample kit following manufacturer's recommended procedure. Briefly, 100 ng of total RNA was first depleted of ribosomal RNA with Ribozero Gold magnetic beads and further purified with Agencourt AMPure XP beads. RNA was fragmented at 94 °C for 8 min and primed with random primer. The fragmented RNA was then converted to double strand cDNA, end repaired, A tailed, and ligated with Unique Dual Indexed adaptors. The adaptor added cDNA library was then PCR amplified using the following conditions: 94 °C for 30 s, 15 cycles of 98 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min. The PCR amplified library was purified using Agencourt AMPure XP magnetic beads and run out on Agilent High Sensitivity DNA Chip for quality check. Individual library was then further quantified using KAPA library quantification qPCR kit and pooled. The pooled library was sequenced on Illumina NovaSeq S1 200cycle kit for 2X100bp sequencing.