Project description:This study began with 72 male 4-week-old BALB/c mice. The mice were split evenly into one of four cohorts: Control, River, Pine, and Road. The control mice were raised with standard corn cob bedding whereas the remaining mice were raised with clean bedding amended with 300 mL of one of three different types of soil. The soil exposure continued throughout the experiment, with 300 mL of new soil added with bi-weekly cage changes. The soils used to amend the cage bedding were previously characterized as having high (Pine), medium (River), and low (Road) diversity. The River and Pine soil were collected from Duke Forest and the Road soil was collected adjacent to Highway 15-501 in Chapel Hill, North Carolina. All mice were given a standard diet and the cages were distributed reverse osmosis treated water through a centralized Lixit® system that was fed to each cage in parallel. After 32 days of standard rearing with amended soils, the mice were exposed via oropharyngeal aspiration to either live influenza A (PR8) virus or heat inactivated (HI) virus.
Project description:Adult male and female C56BL6J mice were subjected to 8 weeks of small mouse cage (SMC) intervention to model physical inactivity. Widely-used physical inactivity models such as hindlimb unloading and immobilization do not phenocopy metabolic adaptations that occur with human sedentary behavior, whereas many aspects of metabolic adaptations with SMC appear to reproduce this effect. The RNAseq analyses were performed in muscles from these mice to capture changes in skeletal muscle gene expression landscape.
Project description:We developed SLIC-CAGE (Super-Low Input Carrier-CAGE) approach to capture 5'end of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. The dramatic increase in sensitivity compared to existing CAGE methods is achieved by specially designed, selectively degradable carrier RNA. We apply SLIC-CAGE on mouse primordial germ cells embryonic day (E) 11.5 - 2 biological replicates.
Project description:We developed SLIC-CAGE (Super-Low Input Carrier-CAGE) approach to capture 5'end of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. The dramatic increase in sensitivity compared to existing CAGE methods is achieved by specially designed, selectively degradable carrier RNA. We tested SLIC-CAGE on Saccharomyces cerevisiae (BY4741 strain) and produced libraries from 1-100 ng of total cellular RNA. We also produced S. cerevisiae nAnT-iCAGE libraries as the current gold-standard CAGE libraries using the recommended 5 micrograms of total cellular RNA to assess the quality of SLIC-CAGE libraries produces with up to 1000-fold less material. We provide a direct comparison between SLIC-CAGE and the latest nanoCAGE protocol (libraries created using S. cerevisiae total RNA) and show that SLIC-CAGE produces unbiased libraries of higher complexity and quality than nanoCAGE. Finally, we provide SLIC-CAGE libraries on mouse embryonic stem cells (E14) using 5-100 ng of total cellular RNA as starting material.
Project description:Scratching damages upper layers of the skin, breaks this first line of immune defence, and leads to inflammation response, which often also modifies the microbiota of the skin. Although the healing of incision wounds is well-described, there are fewer studies on superficial wounds. We used a simulated model of skin scratching to study changes in the host transcriptome, skin microbiota, and their relationship. Additionally, we examined the effect of nanosized ZnO, TiO2, and Ag on both intact and damaged skin. At 24 h after exposure, the number of neutrophils was increased, 396 genes were differentially expressed, and microbiota compositions changed between scratched and intact control skin. At 7 d, the skin was still colonised by gut-associated microbes, including Lachnospiraceae, present in the cage environment, while the transcriptomic responses decreased. To sum up, the nanomaterial exposures reduced the relative abundance of cutaneous microbes on healthy skin, but the effect of scratching was more significant for the transcriptome than the nanomaterial exposure both at 24 h and 7 d. We conclude that superficial skin scratching induces inflammatory cell accumulation and changes in gene expression especially at 24 h, while the changes in the microbiota last at least 7 days.
Project description:Super-Low Input Carrier Cap Analysis of Gene Expression (SLIC-CAGE) to identify transcription start sites (TSS) and existing genome-wide maps of islet histone marks to characterise the contribution of transcriptional regulation of LKB1-mediated control of gene expression in mouse β-cells. SLIC-CAGE was performed as in (Cvetesic et al. - doi: 10.1101/gr.235937.118) using 100 ng of total RNA extracted as described above from islets isolated from a 18 week old female mouse on a C57BL6/J background. 12 cycles were performed for library amplification. The amplified library was purified with AMPure XP beads and visualised using a HS DNA chip (Bioanalyzer, Agilent). The library was sequenced on a HiSeq2500 instrument with paired-end, 150bp reads). SLIC-CAGE paired-end sequencing data was aligned to GENCODE assembly annotation version GRCm38.p6 using the STAR alignment tool v2.4.2a.
Project description:The mammalian sex-determining gene Sry induces male development. Through analyzing transcriptome of pre-Sertoli cells, we identified a novel sequence transcribed within palindromic sequence surrounding Sry. To examine whether this sequence contains transcription start site, Cap analysis gene expression (CAGE) -seq analysis was performed, in which putative transcriptional start sites can be identified by sequencing the 3’ end of cDNA corresponding to 5’ end of RNA. CAGE seq revealed that Cap site was absent within the novel sequence, whereas Sry transcription start site found to locate in minus strand at chrY:2,663,800-2,663,900. Combining the result from long-read RNA seq, we concluded that this sequence is the second exon of mouse Sry.
Project description:5'Cap-Analysis of Gene Expression (5' CAGE) from mesodermal and whole embryo RNA at three different time intervals during development.
Project description:SLIC-CAGE (Super-Low Input Carrier-CAGE) development: comparison with Saccharomyces cerevisiae nAnTi-CAGE and nanoCAGE libraries and validation of SLIC-CAGE on Mus musculus total RNA
