GSM9369082: E. coli BW25113 bacterial cells, V2, SS, GE, Bio Rep 2; Escherichia coli; ncRNA-Seq
The isolation of small RNAs from bacterial cells was performed using the PureLink miRNA Isolation Kit with slight modifications. Cells were lysed using TRIzol (Sigma-Aldrich), followed by chloroform treatment and centrifugation to separate the RNA-containing aqueous phase. Total RNAs were passed through Spin Cartridges with different ethanol concentrations to selectively bind and separate large and small RNAs. Finally, the small RNAs were eluted in RNase-free water after washing and a final centrifugation step. cDNA libraries were prepared using 75 ng of small RNAs as the input, following four variations of the AQRNA-seq library constuction protocol. Variant 1 represents the original protocol initially published by Hu et al. (2021) and later elaborated by Chen et al. (2024), while variants 2, 3, and 4 involve alternative adaptations of the original protocol with respect to the approaches for mitigating polymerase fall-off and/or primer dimers. A side-by-side comparison across the protocols represents the focus of this study.