Next-generation sequencing (NGS)

RNA samples from the circulating exosomes of three mice with CLP were pooled for next-generation sequencing (NGS). The exosomal RNA samples from three mice without CLP (sham) were pooled for use as a control. The RNA samples were sent to GeneTech Biotech Co., Ltd. (Taipei, Taiwan) for cloning. The miRNA population, with lengths of 15-30 nucleotides (nt), was passively eluted from polyacrylamide gels, precipitated with ethanol, and dissolved in water. Linkers were ligated to the small RNAs, and bar-coded cDNAs were prepared using the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, Adapters were ligated to the 3′ and 5′ ends of an aliquot (1 μg) of the pooled small RNAs. Then, adapter-ligated RNAs were reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and amplified by polymerase chain reaction (PCR) in 15 cycles. The samples were indexed with barcodes of 15 variants of the reverse primers. A barcode was ligated directly to the miRNA to significantly reduce sample bias. Individual libraries were analyzed on a BioAnalyzer (Agilent Technologies) for the presence of linked cDNA, and 11 bar-coded libraries of the appropriate size (135-165 bp) were generated.