Project description:Lungs were collected as soon as possible after thermal injury 30-40 mins post injury for neutrophil isolation. Lungs were minced in HBSS with 1% BSA (Sigma) and strained through a 40um nylon filter (BD). Samples were sorted using BD fusion machine. A 100um nozzle was used at a flow rate of 2.RNA was extracted from these neutrophils and microarray was performed.
Project description:This data was used as an example to illustrate a computational method for assessing statistical significance in microarray experiments Contributed by 'The Inflammation and the Host Response to Injury Collaborative Research Program.' Keywords: Two group comparison Genomic response one day post traumatic injury was compared between patients having early or late respiratory recovery
Project description:To compare the transcriptome of ASCs and FAPs before and after injury, we performed quadriceps injury using glycerol in mice. We then isolated subcutaneous adipose tissue and quadriceps muscle and isolated ASCs and FAPs respectively (3 mice per condition). We then extracted RNA and performed RNA-seq.
Project description:This data was used as an example to illustrate a computational method for assessing statistical significance in microarray experiments Contributed by 'The Inflammation and the Host Response to Injury Collaborative Research Program.' Keywords: Two group comparison
Project description:The injury response in the skin is highly complex and involves input from multiple cell types. Single nuclei RNA sequencing was performed to broadly characterize changes in gene expression from all skin cell types early after injury compared to the unwounded state.
Project description:The purpose of the experiment was to determine whole transcriptome changes after spinal cord injury (SCI) to understand how photobiomodulation promotes neuroprotection and functional recovery. SCI was performed at the thoracic (T) level T8, by crushing the dorsal columns using a calibrated watchmaker's forceps. Animals were then treated with photobiomodulation (PBM; 660nm wavelength) or sham control light, within 15 minutes of the injury. PBM or sham-treatment was treatment directed at the lesion site for 1 minute duration every 24hr for the first 3 days. Animals were then killed, tissues harvested and the RNA was extracted using RNEasy lipid tissue mini kit (Qiagen) according to the manufacture's instructions. Whole genome sequencing was outsourced to Qiagen RNA Sequencing Services (Germany).Library preparation was performed using the QIAseq Stranded Total RNA Library Kit with QIAseq FastSelect rRNA and globin depletion. QIAseq FastSelect rRNA HMR was used to reduce the amount of unwanted RNA species. After first and second strand synthesis, the cDNA was end-repaired and 3’ adenylated. Sequencing adapters were ligated to the overhangs. Adapted molecules were enriched using 16 cycles of PCR and purified by a bead-based cleanup. Library preparation was quality controlled using capillary electrophoresis (High Sensitivity Tape D1000) and high-quality libraries were pooled based on equimolar concentrations. The library pool(s) were quantified using qPCR and optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing on a NovaSeq (Illumina Inc., Madison, USA) instrument (2x75, 2x10) according to the manufacturer instructions (Illumina Inc.). Raw data was de-multiplexed and FASTQ files for each sample were generated using the bcl2fastq software v2.20.0.422 (Illumina inc.).
Project description:Skeletal muscle possesses a remarkable capacity to regenerate when injured, but when confronted with major traumatic injury resulting in volumetric muscle loss (VML), the regenerative process consistently fails. The loss of muscle tissue and function from VML injury has prompted development of a suite of therapeutic approaches but these strategies have proceeded without a comprehensive understanding of the molecular landscape that drives the injury response. Herein, we administered a VML injury in an established rodent model and monitored the evolution of the healing phenomenology over multiple time points using muscle function testing, histology, and expression profiling by RNA sequencing. The injury response was then compared to a regenerative medicine treatment using orthotopic transplantation of autologous minced muscle grafts (~1 mm3 tissue fragments). A chronic inflammatory and fibrotic response was observed at all time points following VML. These results suggest that the pathological response to VML injury during the acute stage of the healing response overwhelms endogenous and therapeutic regenerative processes. Overall, the data presented delineate key molecular characteristics of the pathobiological response to VML injury that are critical effectors of effective regenerative treatment paradigms.
