RNA-Seq of Staphylococcus xylosus: Colony cells
The sequencing library was constructed as follows: Cells from Staphylococcus xylosus colonies were harvested and immediately snap-frozen in liquid nitrogen to preserve RNA integrity. Total RNA was extracted using a commercial RNA extraction kit (e.g., Qiagen RNeasy Mini Kit), which includes a DNase I digestion step to remove genomic DNA contamination. The quality and quantity of the extracted RNA were assessed using a spectrophotometer (measuring absorbance at 260/280 and 260/230 ratios) and an Agilent Bioanalyzer to ensure high integrity (RNA Integrity Number, RIN  7).For mRNA enrichment, poly(A)-tailed RNAs were isolated using oligo(dT) magnetic beads, followed by fragmentation of the selected RNAs into small pieces. Subsequently, first-strand cDNA was synthesized using reverse transcriptase and random hexamer primers, and second-strand cDNA was generated through DNA polymerase - mediated reactions. The double-stranded cDNA was then end-repaired, A-tailed, and adaptors were ligated to both ends. The final library was amplified by PCR to enrich the adapter-ligated cDNA fragments, and the amplified library was purified using magnetic beads. The quality and size distribution of the constructed library were verified using the Agilent Bioanalyzer before sequencing on an Illumina NovaSeq platform with paired-end 150bp reads.
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