GSM8881410: pLG008-SP15-1; Escherichia coli str. K-12 substr. DH10B; ncRNA-Seq
Total RNA was extracted using miRNA isolation kit (QIAGEN, catalog number 217004), according to the manufacturer's recommendation. Over 2g total RNA was resolved on Urea-PAGE and recovered within a size window of 60-100 nt for tRNA. Thereafter, purified tRNA was demethylated, alkaline-hydrolyzed by carbonate buffer, dephosphorylated by calf intestinal phosphatase (CIP), and re-phosphorylated by T4 polynucleotide kinase (T4 PNK). Total RNA sample integrity was initially assessed using agarose electrophoresis, and RNA concentrations were measured using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). Total RNA samples underwent pretreatment to remove RNA modifications that could interfere with small RNA sequencing library preparation. Specifically, pretreatment steps included: deacylation of 3'-aminoacyl (charged) RNA to 3'-OH for 3'-adapter ligation, removal of 3'-cP (2',3'-cyclic phosphate) to generate 3'-OH for 3'-adapter ligation, phosphorylation of 5'-OH (hydroxyl group) to 5'-P for 5'-adapter ligation, and demethylation of m1A and m3C for efficient reverse transcription.  Following pretreatment, the total RNA was utilized for the preparation of tRF and tiRNA sequencing libraries. Library preparation involved a sequential workflow: (1) 3'-adapter ligation, (2) 5'-adapter ligation, (3) cDNA synthesis, (4) PCR amplification, and (5) size selection of PCR products between 134–160 bp, corresponding to 14–40 nt small RNAs. The libraries were analyzed and quantified using the Agilent 2100 Bioanalyzer (Agilent, USA). Equal amounts of each library, based on the quantification results, were pooled for sequencing. Sequencing was performed using an Illumina sequencer. Pooled libraries were denatured with 0.1 M NaOH to produce single-stranded DNA molecules and diluted to a final concentration of 1.8 pM. The libraries were loaded onto the sequencing reagent cartridge, and sequencing runs were conducted following the manufacturer's protocols. The NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs, USA, Catalog number# E7300L) was used for library construction.