Inducing the Prophage Lytic Replication for Transcriptomic Analysis

Begin with a bacterial culture carrying an integrated prophage genome repressed by a phage repressor protein, maintaining a dormant lysogenic phase.

Add an inducer and incubate with shaking to trigger cellular stress.

The cellular stress induces autocleavage of the repressor protein, allowing the prophage to transition from lysogeny to lytic replication.

The prophage DNA excises from the bacterial genome and sequentially transcribes early regulatory genes, middle DNA replication genes, and late structural and lysis genes.

Transfer the culture to a large volume of medium to dilute the inducer and incubate to allow cell recovery.

At defined intervals, add a stop solution to arrest transcription and preserve the RNA profile in the bacterial cells.

Centrifuge the culture and discard the supernatant. Resuspend the cells, transfer the suspension to a small tube, and centrifuge.

Remove the supernatant and flash-freeze the pellet to maintain RNA integrity.

Finally, add an RNA-extraction reagent and homogenize to disrupt the cells. The sample is ready for RNA isolation and subsequent transcriptomic analysis.

Next, add norfloxacin at a final concentration of one microgram per milliliter to the induce