The Seibold Laboratory is an 1800 square-foot facility located in the Smith Building on the National Jewish Health Campus in Denver, CO. The Smith Building opened in 2007 and is a modern clinical and research space which is the home of the Center for Genes, Environment and Health (CGEH) investigators.
The Seibold Laboratory is formed by integration of both a state-of-the-art "wet lab," where a range of cellular and molecular studies are performed, and a computational "dry lab," analyzing genome and population scale data sets.
To serve the needs of our research projects and collaborators, the Seibold Laboratory houses the instrumentation and expertise to perform a diverse array of experimental and computational analyses, some of which we highlight below...
3-DIMENSIONAL HUMAN PRIMARY CELL MODELS
A major focus of our laboratory is based on in vitro experimentation using fully functioning muco-ciliary airway epithelial cultures. We generate these cultures from airway epithelial stem cells (basal cells) we isolate from donor airway specimens across various studies. These muco-ciliary epithelial cultures allow us to study how the airway epithelium functions and responds to disease risk factors like viruses, air pollution particulate matter, inflammation, and cigarette smoke in a controlled laboratory setting. Moreover, we are performing these studies on cultures generated from hundreds of donors, from whom we have also collected genetic data, allowing us to study gene x environment interactions at the level of airway cells.
ROBOTIC NEXT GENERATION SEQUENCING LIBRARY PREPARATION
The Seibold Lab operates and maintains a Beckman Coulter Biomek FXP Dual Multichannel 8 span robot with BFX 96-channel head capable of all RNA sample normalization, 96- & 384-well qPCR and molecular biology reaction setup, barcoded adapter stamping, and a range of Next Generation library preparation protocols. In particular the Seibold Lab has developed Biomek protocols for automated generation of targeted DNA sequencing, targeted RNA sequencing, and whole transcriptome sequencing libraries in 96 well format.
PROCESSING AND BIOBANKING HUMAN
AIRWAY AND LUNG TISSUE
As part of the Human Live Cell Core at NJH, we continuously receive and process human lungs and tracheas from deceased donors as well as bronchial and nasal airway epithelial brushings from consented patients, which we incorporate into our studies. In particular, we isolate airway epithelial cells from these specimens for both molecular analyses and primary cell culture. We also process and fix tracheal and lung airway tissue for histological examination in our research studies.
SINGLE CELL RNA SEQUENCING AND ANALYSIS
The Seibold laboratory operates and maintains a Wafergen ICELL8 single cell sequencing instrument capable of processing 1800 cells in a single setting for single cell RNA sequencing library generation. Additionally, through the NJH CGEH the Seibold Lab has access to the 10X Genomics Chromium controller capable of generating single cell RNA-seq libraries on 500-10,000 cells per sample. Our lab has optimized experimental single cell protocols on these platforms for a range of airway, lung, and immune cell samples.
AIRWAY EPITHELIAL CRISPR-CAS9 GENE EDITING
The development of CRISPR-Cas9 technology has revolutionized the ability of scientists to alter the genome of human cells. While this technology is promising, it's application to any particular human cell type or tissue is difficult and requires extensive development. We have developed a protocol to robustly “knock-out” genes in primary airway epithelial cells. We use these knockout cells to determine the function of genes that have been associated with the development of asthma and other lung diseases. We continue to develop protocols to manipulate gene and genetic variant function in airway epithelial cells in our lab and as part of the REGEN program.
BIOINFORMATICS, STATISTICAL GENETICS, AND COMPUTATIONAL BIOLOGY
The computational/statistical side of the Seibold Lab has office space on the 6th floor of the Smith Building adjacent to the Seibold wet lab. The Seibold Lab is a primary user of the NJH High Performance Linux Computing Cluster (funded by S10 RR031832-01). This cluster has 66 Xeon X5660 2.67 GHz, 8 Xeon E7540 2.00GHz, 8 Xeon E5-2630 2.00GHz and 8 Xeon E5-2650 2.00GHz compute nodes for a combined total of 556 available cores and 1112 computing threads and a combined total of 3032GB RAM, 83 TB local storage, and 368 TB networked storage. There is an array of genetic and genomics analysis programs installed and in working condition on the cluster. In particular, the Seibold Lab has developed analytical pipelines and workflows to perform a range of RNA-seq, DNA-seq, and single cell RNA-seq analyses, including QC, mapping, gene/transcript quantitation, variant calling, and network, systems, and machine learning analyses. Additionally, the Seibold lab has developed pipelines for integrative genetic analyses including expression quantitative trait (eQTL) and allelic imbalance, as well as viral metagenomics analyses. The Seibold Lab is also well-versed in a range of genetic analyses including genome wide association studies (GWAS).