Dna 2500 kit

The RNA library for next-generation sequencing (NGS) was constructed with VAHTS Universal V8 RNA-seq Library Prep Kit (Vazyme Biotech, China) by using a total of 10⁹ copies of SARS-CoV-2 RNA; the copy number was predetermined with a COVID-19 Multiplex 1-Step RTqPCR Kit (Topgen Biotech, Taiwan). In brief, the RNA was pretreated with divalent cations at 94°C for 8 min to obtain small RNA pieces with a length of 150–200 nucleotides. Next, the small pieces of RNA were reverse-transcribed following the manufacturer’s procedures to construct a paired-end cDNA library with an average insert size of approximately 150 bp. The cDNAs were ligated to barcode sequencing adapters, and the quality of the cDNA library was analyzed using a MultiNA MCE-202 (Shimadzu, Japan) with a DNA 2500 Kit (Shimadzu, Japan). NGS of the paired-end cDNA library was performed using a NovaSeq 6000 Sequencing System (Illumina, United States) following the manufacturer’s standard protocol. Approximately twenty million paired-end reads (∼150 bp per read) were produced per cDNA library using a paired-end RNA-seq approach (Illumina, United States). The adapter sequences were trimmed from the sequence reads and filtered by using fastp (v 0.19.5) (33 (link)) with a quality value (QV) ≥ 20; the read lengths were filtered by Filter FASTQ (v1.1.5) (34 (link)) with a cut-off ≥145 bp.

A total of 10 ng exomiRs were used for library preparation, following the manual of VAHTS Small RNA Index Primer Kit (Vazyme, NR801, China). The correct barcoding adapter-ligated library amplicon length will be around 142 bp and amplicons were purified using FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech, CN). The concentration was quantified using microfluidic electrophoresis and the length of the eluted barcoding adaptor-ligated library was checked by DNA-2500 Kit on MultiNA MCE-202 (Shimadzu, Japan). The qualified pooled library (4nM) was sequenced on an Illumina NextSeq 500 (single-end, 75 sequencing cycles) (Illumina, USA). NGS was performed twice, and 14 most abundance of miRNAs in conditioned PFTSC (Table 2) were selected to further analysis and experiments.