Project description:We used ATLAS-seq-neo to map the sites of integration of an engineered LINE-1 (L1) retrotransposon into the genome of HeLa S3 cells. In brief, we transfected cells with a plasmid-borne L1.3 element carrying a neomycin-resistance-based retrotransposition cassette, as well as a hygromycin-resistance cassette on the plasmid backbone. For this set of experiments, cells were only selected for transfection (hygromycin) but not for retrotransposition (neomycin). Then we prepared ATLAS-seq-neo libraries. Each sample corresponds to an independent transfection and pool of hygromycin-resistant cells. ATLAS-seq-neo relies on the random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence, and Ion Torrent sequencing using single-end 400 bp read chemistry. The primer used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016). For some libraries, the linker-ligated genomic DNA was digested with BamHI, which cuts downstream of L1 polyA site in the plasmid backbone, to limit amplification from the plasmid and enrich for retrotransposition-mediated insertion events into the genomic DNA.
Project description:We used ATLAS-seq-neo to map the sites of integration of an engineered LINE-1 (L1) retrotransposon into the genome of HeLa S3 cells. In brief, we transfected cells with a plasmid-borne L1.3 element carrying a NeoR-based retrotransposition cassette. Cells were selected by G418 and used to prepare ATLAS-seq-neo libraries. Each sample corresponds to an independent transfection and pool of G418-resistant cells. ATLAS-seq-neo relies on the random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence, and Ion Torrent sequencing using single-end 400 bp read chemistry. The primer used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016).
Project description:We used ATLAS-seq to map the sites of integration of an engineered LINE-1 (L1) retrotransposon into the genome of HeLa S3 cells. Then, we compared the position of these sites with publicly available genomic datasets. In order to cross-corroborate our findings with datasets obtained in the same cell stock as used in our retrotransposition assays, we also performed H3K4me1 ChIP-seq.
Project description:Damage that affects large volumes of skeletal muscle tissue can severely impact health, mobility, and quality-of-life. Efforts to restore muscle function by implanting engineered grafts at the site of damage have demonstrated limited restoration of force production. Various forms of mechanical and biochemical stimulation have been shown to have a potentially beneficial impact on muscle maturation, vascularization, and innervation, but yield unpredictable and inconsistent recovery of functional mobility. Here we show that targeted exercise of optogenetic engineered muscle grafts restores motor functions 2 weeks post-injury. Furthermore, we conduct phosphoproteomic analysis of grafts in vitro and in vivo to show that exercise training alters signaling pathways that play key roles in skeletal muscle contractility, neurite growth, neuromuscular synapse formation, angiogenesis, and cytoskeletal remodeling. Our study uncovers several proteins not previously known to be modulated by exercise, revealing promising mechanisms for leveraging targeted exercise to enhance functional integration of tissue engineered muscle.
Project description:We performed RNA sequencing on HeLa cells treated with OGT inhibitor (Ac4-5SGlcNAc), DMSO, or OGA inhibitor (Thiamet-G) and obtained about 100 million paired-end reads (150 bp) for each replicate. Then the sequencing data was analyzed by MISO to select the O-GlcNAc-regulated alternative splicing events.
Project description:We report a comprehensive analysis of alterations in gene expression and alternative splicing upon RBM39 depletion and rescue in HeLa cells.