ScRNA-seq of Neutrophils upon acute lung injury in a biomaterial-induced chronic inflammatory mouse model
Mice were first anesthetized using high dose ketamine-xylazine solution. After anesthesia, about 500-1000 μL blood was collected through the retro-orbital vein. Following euthanasia, bronchoalveolar lavage fluid (BALF) was collected using a 20-G cannula was inserted into the trachea and fixed with a 93 ligature. Lungs were then lavaged with 1 mL sterile Ice-cold 1X phosphate buffered saline (PBS) two times (total volume 2 mL) to collect BALF. Fluid recovery was >85% each time. Blood and BALF samples were then centrifuged at 400 RCF for 5 mins at 4°C. The supernatant was stored at -80°C and the cell pellet was subjected to RBC lysis in ACK Lysis buffer and resuspended in 1mL PBS for counting using a hemacytometer. 1) Biomaterial implantation: Chitosan microspheres (337± 47 micrometers) were prepared using the Spraybase® Electrospray system inside a sterile biosafety cabinet and chemically sterilized as described22. Microspheres were surgically implanted into the peritoneal cavity of mice to induce chronic inflammation. In brief, microspheres (450 µL) were suspended in 450 µL sterile saline for implantation. The entire volume (900 µL) was then implanted into the peritoneal space22,27 following a laparotomy procedure. In controls (also called mock-surgery), the animals went through the surgical procedure and were injected with 900 µL sterile saline.  2) Acute Lung Injury: Mice were anesthetized with ketamine (120 mg/kg) and xylazine (40 mg/kg) and placed on a board at an angle of 50 degrees in a supine position. The tongue was carefully pulled using sterile forceps, and the chest was illuminated using a cold source light to visualize the tracheal opening. IV cannula (22G) was inserted into the trachea. LPS (0.4 mg/kg, Escherichia coli O127:B8; Sigma-Aldrich, Merck) was administered through the cannula in 25 μL of sterile saline using a 200 μL pipette tip. The mice were gently massaged and allowed to recover on a heating pad. Immune cells from blood and BALF were isolated as described in earlier sections. Sample tagging was performed by labeling cells with BD® Mouse Single-Cell Multiplexing Kit. Neutrophils in the samples were tagged with Ly6G antibody-derived tags (Ly6G-ADTs, BD® AbSeq Ab-Oligos). These cells were subjected to single cell capture and cDNA Synthesis, library construction using a Whole Transcriptome Analysis (WTA, BD) kit with the BD Rhapsody™ single-cell analysis system following standard procedures provided by the manufacturer Sample tagging was performed by labeling cells with BD® Mouse Single-Cell Multiplexing Kit. Neutrophils in the samples were tagged with Ly6G antibody-derived tags (Ly6G-ADTs, BD® AbSeq Ab-Oligos). These cells were subjected to single cell capture and cDNA Synthesis, library construction using a Whole Transcriptome Analysis (WTA, BD) kit with the BD Rhapsody™ single-cell analysis system following standard procedures provided by the manufacturer.