WT, replicate1
An overnight culture of the oda mutant was grown anaerobically in TSPC medium supplemented with 20 mM putrescine. The following day, cells were harvested by centrifugation, washed twice with fresh TSPC medium to remove residual putrescine, and resuspended in fresh TSPC at an OD of 1.0. Cultures were then subcultured at a 1:20 dilution into TSPC medium containing either 20 mM or 0.25 mM putrescine and incubated anaerobically. After 10 hours of growth, cells were collected for RNA extraction. A 10-hour wild-type culture grown without putrescine was also prepared for comparison. RNA samples were submitted to Cancer Genomics Center at The University of Texas Health Science Center at Houston (CPRITRP240610). Total RNA was quality-checked using Agilent RNA 6000 Pico kit (#5067-1513) by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA). RNA with an integrity number of greater than 7 was used for library preparation. Libraries were prepared from the rRNA depleted RNAs withNEBNext Ultra II Directional RNA Library Prep Kit for ILLumina (E7760L, New England Biolabs) and NEBNext Multiplex Oligos for illumina (E6609S, New England Biolabs) following the manufacturers instructions. The quality of the final libraries was examined using Agilent High Sensitive DNA Kit (#5067-4626) by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA) , and the library concentrations were determined by qPCR using Collibri Library Quantification kit (#A38524500, Thermo Fisher Scientific). Before 2024, The libraries were pooled evenly and went for the paired-end 75-cycle sequencing on an Illumina NextSeq 550 System (Illumina, Inc., USA) using High Output Kit v2.5 (#20024907, Illumina, Inc., USA). Since 2024, ). The libraries were pooled evenly and went for the paired-end 150-cycle sequencing on an Illumina NovaSeq System (Illumina, Inc., USA).