title: GSM8841553: Pseudomonas donghuensis HYS fur mutant, growth on NGM plate, 24h; Pseudomonas donghuensis; RNA-Seq
library_construction_protocol: For the Caenorhabditis elegans  samples, collected nematodes were ground rapidly from liquid nitrogen and processed using an RNA extraction kit (Nanjing, Vazyme, RC112). Ground nematodes were resuspended in lysis buffer, added to a genomic DNA removal column, centrifuged at 12,000 rpm for 1 minute, filtrate collected, and RNA adsorbed, washed twice, and eluted using RNase-free water. For bacteria samples, mRNA was extracted using the Trizol method. Following the lysis of bacterial cells, guanidine isothiocyanate in Trizol was employed to denature proteins and inhibit RNase activity. RNA was then separated from the aqueous phase through chloroform partitioning, and total RNA (including mRNA) was precipitated with isopropanol. Subsequently, impurities were removed by ethanol washing, and the final product was dissolved in nuclease-free water. For the Caenorhabditis elegans samples, including the Dual-RNA seq samples, the enriched mRNA consisted of complete RNA sequences, which were randomly fragmented by adding fragmentation buffer to generate small fragments of approximately 300 bp. Under the action of reverse transcriptase, random primers were utilized to synthesize a single strand of cDNA using mRNA as the template, followed by the synthesis of the second strand to form a stable double-stranded structure. The double-stranded cDNA structure featured sticky ends, which were converted to blunt ends by adding End Repair Mix. Subsequently, an A base was added to the 3' end, and the products were purified and size-selected after adapter ligation, followed by PCR amplification of the selected products to obtain the final library. For the bacterial samples, the TruSeq™ Stranded Total RNA Library Prep Kit was employed to construct the library. In the synthesis of the second strand of cDNA, dUTP was used in place of dTTP in the dNTP reagent, resulting in the incorporation of A/U/C/G bases in the second strand of cDNA. Prior to PCR amplification, UNG enzyme was utilized to digest the second strand of cDNA, ensuring that the library contained only the first strand of cDNA.