Nextseq 550dx system

Total RNA purification from cell cultures was performed by miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA concentration was determined with a NanoDropOne spectrophotometer (Thermo Fisher, Waltham, MA, USA) and its quality was assessed with the TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA). Indexed libraries were prepared from 500 ng/each purified RNA using the NEXTFLEX Small RNA-Seq Kit v3 (PerkinElmer). Libraries were quantified with the TapeStation 4200 (Agilent Technologies) and Qubit fluorometer (Invitrogen Co.), then pooled such that each index-tagged sample was present in equimolar amounts. The pooled samples were then subjected to cluster generation and sequencing using an Illumina NextSeq 550 Dx System (Illumina) in a 1 × 75 single-end format. The generated raw sequence files (.fastq files) underwent quality control analysis using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (accessed on 22 September 2022). The sRNAbench tool [23 (link)] was then used to remove adapter sequences and low-quality reads to obtain the miRNA expression profiles. Finally, the Bioconductor package DESeq2 [24 (link)] in R was used to normalize the data using the median of ratios method, and to perform the differential expression analysis between the various experimental condition.

The miRNA profile of circulating EVs in breast cancer patients was performed by RNA-seq. Indexed libraries were prepared with NEXTFLEX Small RNA-Seq Kit v3 (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instructions. Libraries were quantified using the TapeStation 4200 (Agilent Technologies) and Qubit Fluorometer (Thermo Fisher Scientific), then pooled such that each index-tagged sample was present in equimolar amounts. The pooled samples were subject to cluster generation and sequencing using an Illumina NextSeq 550 Dx System (Illumina, San Diego, CA, USA) in a 1 × 75 single-end format. The raw sequencing files are deposited in the NCBI’s Gene Expression Omnibus and are available through GEO Series accession number GSE222681.

Genomic DNA samples extracted from the peripheral blood lymphocytes of each patient were used for library preparation and target capture with a custom panel that targeted candidate genes. Massively parallel sequencing was performed on the NextSeq 550Dx System (Illumina; San Diego, CA, USA). GRCg37 (hg 19) was used as the reference sequence for mapping and variant calling. Databases used for analysis and variant annotation include the Online Mendelian Inheritance in Man (OMIM), the Human Gene Mutation Database (HGMD), ClinVar, dbSNP, the 1000 Genome Project, the Exome Aggregation Consortium (ExAC), the Exome Sequencing Project (ESP), and the Korean Reference Genome Database (KRGDB). The classification of variants followed the standards and guidelines established by the American College of Medical Genetics (ACMG). All pathogenic and likely pathogenic variants were further confirmed by Sanger sequencing.

First, DNA and RNA were extracted using Magnetic serum/plasma DNA Maxi kit (Tiangen Biotech (Beijing) Co. Ltd., China). For BALF specimens, HostZERQ Microbial DNA kit (Jianshi Biotech (Beijing) Co. Ltd., China) was used to remove human nucleic acids for further nucleic acid extraction. Second, nucleic acids were fragmented into 150–300 bp in length with Bioruptor (Diagenode Diagnostics, Belgium) that is a non-contact ultrasonic disruptor. The library was constructed using Library Preparation kit (Kapabio System, Boston, MA). Third, high-throughput sequencing was conducted with Illumina Next Seq550Dx system (Illumina Inc., San Diego, CA). In the process of sequencing, adaptors, reads with low quality and repeated sequences, and short reads < 36 bp in length were removed. Microorganisms were then identified in the specimens through sequence alignment in the microbial genome database (bacteria, viruses, fungi and parasites) by using Bowtie2 (version 2.3.5) (Genoxor Medical Science and Technology Inc., Shanghai) [24 (link)].