Nmes1 is a novel early regulator of IL-4-induced response in macrophages and regulates the wound healing capacity of the damaged intestine
Nmes1 is a novel early regulator of IL-4-induced response in macrophages and regulates the wound healing capacity of the damaged intestine
To isolate lamina propria cells, colons were removed from mice, cut open longitudinally, and washed in PBS, cut into 4 to 5 smaller pieces, then incubated in 20 mL HBSS containing 200 µL EDTA 0.5 M per sample at 37 °C in a shaker for 20 min to detach epithelial cells. After this first digest, the colon pieces were washed in PBS 3-5 times to remove the epithelial cells. The remaining pieces were then minced to disrupt the tissue and digested in 5 mL HBSS containing 2 % FCS, 3 µL DNaseI and 5 mg collagenase VIII per sample for 45 min at 37 °C on a shaker. After digestion, the suspension was mashed through a 100 µm cell strainer, then filtered again through a 40 µm cell strainer. The filtered suspension was centrifuged at 300 x g for 5 min at 4 °C to pellet the cells, which were then used for further applications. Samples were first lysed using the QiaShredder Kit (Qiagen), and RNA was isolated using the RNeasy Mini Kit (Qiagen).  Library preparation and transcriptome sequencing was performed using 100base/paired-end reads on BGI's DNBSEQTM Technology Platform.