Project description:Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcription program essential for bone formation through both genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed. By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation: proliferation, matrix deposition and mineralization, we identified Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) over the course of these stages, we discovered close to 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibited distinct patterns during osteogenesis, and were associated with proximal promoters as well as a large percentage of Runx2 occupancy in non-promoter regions: upstream, introns, exons, transcription termination site (TTS) regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identified novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of extracellular matrix. We demonstrated by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions. Our data establish that Runx2 interactions withM-BM- chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis. To identiy the genome-wide occupancy of Runx2, DNA bound by Runx2 at the prolieration, matrix deposition, and mineralization stages were recovered by Runx2 ChIP. Libraries of purified DNA were generated using Illumina SR adapters (Illumina) following manufacturerM-bM-^@M-^Ys manual, and were selected for the inserted fragments of 200 M-BM-1 50 bp, and sequenced 36 bases on an Illumina Genome Analyzer II. Base calls and sequence reads were generated by Illumina CASAVA software (version 1.6, Illumina). Two independent biological repeats of Runx2 ChIP-Seq libraries were prepared for each time point, and two input libraries were prepared with sonicated DNA from day 9 MC3T3-E1 cells. We pooled the reads from two biological replicates for peaking calling using MACS (version 1.4.1) with a stringent p value threshold (p < 1e-10) in contrast to input control, and used these peaks for further bioinformatic analyses. Each sample deposited here contains three files: the sequence file with short reads combined from two biological replicates, a Bed file with peaks called from the pooled short reads, and a Wig file with peak signals.
Project description:Purpose: The goal of this study is to compare endothelial small RNA transcriptome to identify the target of OASL under basal or stimulated conditions by utilizing miRNA-seq. Methods: Endothelial miRNA profilies of siCTL or siOASL transfected HUVECs were generated by illumina sequencing method, in duplicate. After sequencing, the raw sequence reads are filtered based on quality. The adapter sequences are also trimmed off the raw sequence reads. rRNA removed reads are sequentially aligned to reference genome (GRCh38) and miRNA prediction is performed by miRDeep2. Results: We identified known miRNA in species (miRDeep2) in the HUVECs transfected with siCTL or siOASL. The expression profile of mature miRNA is used to analyze differentially expressed miRNA(DE miRNA). Conclusions: Our study represents the first analysis of endothelial miRNA profiles affected by OASL knockdown with biologic replicates.
Project description:A cDNA library was constructed by Novogene (CA, USA) using a Small RNA Sample Pre Kit, and Illumina sequencing was conducted according to company workflow, using 20 million reads. Raw data were filtered for quality as determined by reads with a quality score > 5, reads containing N < 10%, no 5' primer contaminants, and reads with a 3' primer and insert tag. The 3' primer sequence was trimmed and reads with a poly A/T/G/C were removed
Project description:Introduction Chlamydia trachomatis (C. trachomatis) is a Gram-negative bacterium and a common human pathogen. The World Health Organization (WHO) estimates that over 130 million people are infected with C. trachomatis globally each year and with increasing incidence. C. trachomatis causes long-lasting and recurrent infections that over time induce severe tissue damage in the female genital tract that can lead to ectopic pregnancy and infertility. Thus, the human immune system fails to control and eradicate C. trachomatis during primary infection and fails to develop protective immunity against secondary infections. In vivo infection models, using complement knock out mice, suggest that the complement system is critically involved in both anti-chlamydial immunity and infection-induced pathology. To increase our understanding of complement-mediated immunity against C. trachomatis we analyzed global complement deposition on serum-incubated C. trachomatis by mass spectrometry. Methods Purified C. trachomatis was incubated in seronegative normal human serum (NHS) or heat-inactivated normal human serum (HI-NHS) for 30 min, thoroughly washed, and processed for mass spectrometry. All samples were lysed, reduced and alkylated and digested with trypsin. Some samples were chemically modified to acetylate free amino groups (N-terminal and lysine amino groups) before trypsin digestion. Peptides were analyzed on a UltimateTM 3500 RSLCnano coupled to a Q Exactive HF-X mass spectrometer. Raw data files were searched against the Uniprot human reference proteome using MaxQuant. Results We demonstrate that C. trachomatis elicits potent complement activation demonstrated by deposition of both early and late complement factors together with several complement regulators. We further demonstrate proteolytically processing of complement C3b to “inactive” C3 cleavage fragments. Conclusion We demonstrate the deposition of several novel complement-associated proteins and -cleavage fragments on the surface of C. trachomatis.
Project description:Whole exome sequencing of 5 HCLc tumor-germline pairs. Genomic DNA from HCLc tumor cells and T-cells for germline was used. Whole exome enrichment was performed with either Agilent SureSelect (50Mb, samples S3G/T, S5G/T, S9G/T) or Roche Nimblegen (44.1Mb, samples S4G/T and S6G/T). The resulting exome libraries were sequenced on the Illumina HiSeq platform with paired-end 100bp reads to an average depth of 120-134x. Bam files were generated using NovoalignMPI (v3.0) to align the raw fastq files to the reference genome sequence (hg19) and picard tools (v1.34) to flag duplicate reads (optical or pcr), unmapped reads, reads mapping to more than one location, and reads failing vendor QC.
Project description:Background: Intramuscular fat (IMF) content is an important index for beef quality. However, the genetics of IMF deposition is complex and still largely unclear, especially in buffalo. To identify miRNAs with potential regulatory role in lipid accumulated in muscle, we performed small RNA sequencing and identified miRNAs expressed in the longissimus dorsi muscle and back fat of Chinese buffalo, which provided vital information for further identification of miRNAs with potential regulatory role in the lipid accumulated in muscle. Results: Three small RNA libraries were constructed. A total of 32762032 raw reads were obtained from adipose groups, respectively. After filtering the adaptor and low quality reads, 32054381 clean reads were retained. In total, 623 miRNAs were identified.
Project description:The centromere, as an essential element to control chromosome segregation, is epigenetically determined by CENH3-containing nucleosomes as a functional marker, therefore the accurate deposition of CENH3 is crucial to chromosome transmission. We characterized the deposition of CENH3 in maize by over-expression and mutational analysis. Our results revealed that over-expressing CENH3 in callus is lethal while over-expressing GFP-CENH3 and CENH3-YFP in callus and plants is not and can be partly deposited normally. Different mutations of GFP-CENH3 demonstrated that CENH3-Thr4 in the N terminus was needed for the deposition as a positive phosphorylation site and the last five amino acids in the C terminus are necessary for deposition. The C terminal tail of CENH3 is confirmed to be responsible for the interaction of CENH3 and histone H4, which indicates that CENH3 maintains deposition in centromeres via interacting with H4 to form stable nucleosomes. For GFP-CENH3 and CENH3-YFP, the fused tags at the termini probably affect the structure of CENH3 and reduce its interaction with other proteins, which in turn could decrease proper deposition. Taken together, multiple amino acids or motifs were shown to play essential roles in CENH3 deposition, which is suggested to be affected by numerous factors in maize.