Hiseq 2500 genome analyzer platform

Transcriptome sequencing of each of the six RNA samples (two different female samples with three biological replicates each) was performed with RNA fragmented to an average of 150 nucleotides. Sequencing was carried out by the Max Planck Genome Centre Cologne (MPGCC) on an Illumina HiSeq2500 Genome Analyzer platform using paired-end (2 × 100 bp) read technology. This yielded ∼25–28 million reads for each of the six samples, respectively. Quality control measures, including the filtering of high-quality reads based on the score given in fastq files, removal of reads containing primer/adaptor sequences and trimming of read length, were carried out using CLC Genomics Workbench v6.5 (http://www.clcbio.com).

Three asthma mice and three SC treatment mice were involved in the RNA sequencing. Total RNA was isolated from BALF-derived exosomes via TRIzol (Invitrogen, Carlsbad, CA, USA). RNA concentration and integrity were measured by the NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). Illumina TruSeq RNA Sample Preparation Kit (illumina, San Diego, CA, USA) was used to establish the small RNA libraries, followed by the manufacturer's recommendations. Briefly, 1 μg of total RNA was adapter-ligated with 120 nt adapter. RNA of target fragment size 135–170 nt was obtained by PAGE gel electrophoresis. Target RNA was collected with anhydrous ethanol and then reversely transcribed into cDNA and quantified with the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). All six libraries were sequenced on an Illumina Hiseq 2500 Genome Analyzer platform using pair-end mode, at Shanghai Yingbio biotechnology co. Ltd.

The smRNA sequencing library was prepared using a TruSeq RNA Sample preparation Kit (Illumina, San Diego, CA, USA), adhering to Illumina’s standard procedure. This preparation involved selecting RNA fractions, ligating adapters, and amplifying the sample to construct the library suitable for high-throughput sequencing. The constructed smRNA library was subsequently sequenced on an Illumina Hiseq 2500 Genome Analyzer platform [26 (link)]. The sequencing parameters were set to achieve a read length of 50 base pairs (bp) using single-end sequencing. This approach was chosen to optimize the detection and analysis of smRNA species, particularly miRNAs. The size range of the library was determined to be between 145 bp and 160 bp, which is indicative of successful library preparation and suitable for effective miRNA sequencing. All processes related to the smRNA library construction and sequencing were performed externally by MACROGEN Inc. (Seoul, Republic of Korea), ensuring high-quality and reliable sequencing data.