Transposon insertion sequencing (TIS) is an emerging technique which utilizes a massive transposon mutant library to screen specific phenotype and determine the conditional essential genetic requirements for bacterial fitness under distinct conditions combined with high-throughput parallel sequencing technology. Compared with a massive mutant library in traditional TIS, the defined mutant library sequencing (DML-Seq) has advantages as: 1) efficient mutagenesis; 2) low bottleneck effects; 3) avoid hotpots caused by screening; 4) can be directly used in the following experiments. Here, we described an optimized procedure of DML-Seq for fitness screen to supply classical TIS using the marine pathogenic bacterium Edwardsiella piscicida as an example.

Decellularized extracellular matrix (ECM) biomaterials derived from native tissues and organs are widely used for tissue engineering and wound repair. To boost their regenerative potential, ECM biomaterials can be functionalized via the immobilization of bioactive molecules. To enable ECM functionalization in a chemoselective manner, we have recently reported an effective approach for labeling native organ ECM with the click chemistry-reactive azide ligand via physiologic post-translational glycosylation. Here, using the rat lung as a model, we provide a detailed protocol for in vivo and ex vivo metabolic azide labeling of the native organ ECM using N-Azidoacetylgalactosamine-tetraacylated (Ac4GalNAz), together with procedures for decellularization and labeling characterization. Our