Transcriptomic analysis of ALPK1-dependent responses in BMDMs from WT and ALPK1 knockout C57BL/6 mice upon ADP-Hep stimulation

The ALPK1 pathway is a cytosolic sensor for the bacterial metabolite ADP-heptose (ADP-Hep), triggering innate immune signalling. To investigate ALPK1-dependent transcriptional responses, bone marrow-derived macrophages (BMDMs) were generated from wild-type (WT) and Alpk1 knockout C57BL/6 mice using L929 cell-conditioned medium. On day 7 of differentiation, BMDMs were treated with either PBS (control) or ADP-Hep. RNA sequencing was performed to characterise gene expression changes, allowing assessment of ALPK1 pathway activation. Overall design: Bone marrow cells were isolated from WT and Alpk1 knockout C57BL/6 mice and differentiated into BMDMs over 7 days in the presence of L929 cell-conditioned medium. Differentiated BMDMs were then treated with PBS (control) or ADP-heptose for 4 hours. Total RNA was extracted from each sample and sequenced to evaluate transcriptional changes. The design allows comparison between PBS and ADP-Hep-treated cells within each genotype, as well as assessment of ALPK1-specific transcriptional responses by comparing WT and Alpk1-/- BMDMs.