Project description:Recently, direct reprogramming between divergent lineages has been achieved by introducing cell-fate-determining transcription factors. This progress may provide alternative cell resources for drug discovery and regenerative medicine. However, the genetic manipulation may limit the future application of these approaches. In this study, we identified a novel small-molecule cocktail that directly converted fibroblasts into neuronal cell fate with a high yield up after 16-days of induction. After a further maturation stage, these chemically-induced neurons (CiNs) possessed neuron-specific expression patterns, generated action potentials and formed functional synapses. Gene expression profiling revealed the activation of neuronal specific genes in the early stage of small molecule treatment. Overall, our findings prove the principle of chemically-induced direct reprogramming of somatic cell fates across germ layers without genetic manipulation, and show that cell fate can be manipulated through disrupting initial cell program and activating target cell master genes with pure chemicals. Total of 15 samples were analyzed, including mouse fibroblasts, mouse cortical primary neurons and chemically-induced neurons by different duration of chemical induction (Day0, Day4, Day8, Day19) and different small-molecule cocktail (FICB, FICB-1)
Project description:The goal of these experiments were to test the on-target and target-adjacent editing efficiencies of different single-nucleobase editing systems. Previous studies have shown that tethering DNA mutating enzymes to Cas9-nickase-UGI complexes results in editing of chromosomal DNA. However, these editing events encompass undesirable target-adjacent nucleobase edits. Here, we characterize a novel approach that reduces the frequency of target-adjacent editing while maintaining a high level of on-target editing.
Project description:We describe the use of a novel DNA modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq) to map high-resolution hydroxymethylome of mouse E14 embryonic stem cells. The specificity of AbaSI enables sensitive detection of 5hmC at low occupancy regions. Bioinformatic analysis suggests 5hmCs in genic regions closely follows the 5mC distribution. 5hmC is generally depleted in CpG islands and only enriched in a small set of repetitive elements. A regularly spaced and oscillating 5hmC pattern was observed at the binding sites of CTCF. 5hmC is enriched at the poised enhancers with the mono-methylated histone H3 lysine 4 (H3K4me1) marks, but not at the active enhancers with the acetylated histone H3 lysine 27 (H3K27Ac) marks. Non-CG hydroxymethylation appears to be prevalent in the mitochondrial genome. We propose that some amounts of transiently stable 5hmCs may indicate a poised epigenetic state or de-methylation intermediate, while others may suggest a locally accessible chromosomal environment to the TET enzymatic apparatus. Mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cell by enzymatic digistion of AbaSI coupled with high-throughput sequencing, in replicates.
Project description:The number of transcripts where allelic bias is dependent parent-of-origin was predicted at 100-200 until two recent studies applied RNA-Seq to brain regions from reciprocally crossed inbred mouse strains and identified over a thousand novel imprinted loci, including hundreds present in only males or females. Reanalysis revealed that the vast majority of these novel loci are explained by technical and biological variation of the approach, and are not genuine cases of general or sex-specific parent-of-origin allelic expression. Independent replication projects that, at most, a few dozen novel imprinted transcripts are present in the dataset, in line with previous projections of 100-200 total imprinted transcripts. Whole brain transcriptome analysis of E17.5 F1 embryos from reciprocally crossed C57BL/6J and CastEi/J parents
Project description:We report the usage of ChIP-mass spectrometry in identifying proteins and histone modifications involved in Drosophila dosage compensation. We identified a chromatin targeting factor, CG4747, that is involved in recognition of H3K36me3 and robust recruitment of the Drosophila MSL complex to its correct targets on the male X chromosome. ChIP-seq with PAP antibody of Drosophila larvae expressing C-terminally TAP-tagged CG4747.
Project description:To study early-onset gene expression changes in cutaneous wound healing, 3 mm wounds were induced into the back skin of female wildtype C57BL/6 mice using a biopsy punch. Mice were sacrificed 2h, 6h or 24h post wound induction (PWI) and 1 - 1.5 mm of skin lining the wound edge was isolated and sequenced. The skin from the initial punch biopsy (0h PWI) was preserved and taken as a control sample to identify differentially expressed genes.
Project description:The data represent whole genome sequencing of two sequential isolates of B. contaminans ST872 that have been retrieved form a cystic fibrosis patient during different phases of chronic pulmonary infection.
Project description:We performed RNA-seq experiments on a total of 12 mouse immune organs, including spleen (SP), bone marrow (BM), lymph node (LN) and Peripheral blood mononuclear cell (PBMC). Briefly, RNA-Seq libraries were constructed after rRNA depletion using a NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB). The E6310L NEBNext Ultra RNA Library Prep Kit for Illumina（NEB, E7530S）(NEB) was used according to the manufacturer’s instructions and the cDNAs were sequenced with the Hiseq X10 platform(Illumina)
Project description:A 2-hour heat-shock at 37C was used to activate hs-FLP and an actin5C-FRT-stop-FRT-GAL4 transgene in larvae carrying any possible combination of the genetic elements UAS-Myc, UAS-Atu-IR, Max-/-. 48 hours later 16-27 wing imaginal discs were isolated from wandering L3 larvae and polyA-RNA was processed for sequencing.
Project description:Purpose: Our previous clinical trials have been demonstrated that Anlotinib can inhibit tumor growth upon refractory advanced non-small cell lung cancer (NSCLC) patients with the possibility mechanism of anti-angiogenesis. The present study sought to reveal the underlying molecular mechanism of Anlotinib-induced anti-angiogenesis in advanced NSCLC. Experimental Design: Computed tomography (CT) was used to evaluate the treatment effect of Anlotinib upon refractory advanced NSCLC patients. Transcriptome profiling was performed to identify the key gene expression alteration in NCI-H1975 cells before and after Anlotinib treatment. NCI-H1975 derived xenograft model was applied to investigate treatment effect and verify anti-angiogenesis mechanism of Anlotinib. Results: Anlotinib induces tumor cytotoxicity on refractory advanced NSCLC patients, NCI-H1975 derived xenograft models and lung adenocarcinoma cell lines. Transcriptome profiling revealed CCL2 blockade could be responsible for Anlotinib-induced anti-angiogenesis. NCI-H1975 derived xenograft model demonstrated Anlotinib-induced CCL2 blockade play an important role in anti-angiogenesis. Conclusions: This study not only offered the first evidence that Anlotinib inhibits angiogenesis via blocking CCL2 expression, but also provided a novel theoretical basis for the application of Anlotinib in advanced NSCLC patients.