title: GSM5100202: E_7; Mus musculus; ncRNA-Seq
library_construction_protocol: The feces were collected once a week and stored at -80°C until processing. Mice were scuffed, and a 1.5 mL Eppendorf tube (Fisher Scientific, Waltham, MA) was placed under the anus to collect 3-5 fecal pellets. If sufficient feces were not collected after 30 seconds, the mouse was placed in a clean cage without beading until at least 3 pellets were obtained. Exosomal RNA was extracted and sequenced as follows. 2 fecal pellets (~60mg) were vortexed with 0.9-2.0 mm stainless steel beads (Next Advance, Troy, NY) with 60 μl Tris-HCl (10 mM) in a 1.5 mL microcentrifuge tube. Increasing amounts of Tris-HCl until 1 mL total volume was reached and subsequently vortexed. The resulting mixture was then centrifuged at 10,000Xg for 5 minutes. The supernatant was then filtered through a 0.2 μm syringe filter (ThermoFisher Scientific, Waltham, MA). Exosomes were isolated via ExoQuick TC (System Biosciences, Palo Alto, CA) according to manufacturer instructions. Purified exosomes were lysed and RNA was isolated using Quick-RNA Microprep Kit (Zymo Research, Irvine, CA). Quality and quantity of the extracted RNA were determined using TapeStation RNA ScreenTape (Agilent Technologies, Santa Clara, CA) and Qubit fluorometer (ThermoFisher Scientific). SmRNA-sequencing libraries were prepared from 50 to 150 ng of total RNA using the QIAseq miRNA Library Kit (Qiagen, Germantown, MD) as per the manufacturer's instructions. This system offers a built-in Unique Molecular Identifier (UMI) application, which is used to eliminate possible PCR duplicates in sequencing datasets and therefore facilitate unbiased gene expression profiling. The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled libraries were diluted to 2 nM in EB buffer (Qiagen) and then denatured using the Illumina protocol. The denatured libraries were loaded onto an SP flow cell on an Illumina NovaSeq 6000 and run for 53-71 cycles using a single-read recipe according to the manufacturer's instructions.