Project description:Total proteome for triplicates of WT and RBPMS KO hESCs to identify differences in proteins abundances due to a loss of RBPMS. Cells were lysed and prepared according to the in solution digest protocol and 50 µg proteins were reduced, alkylated and digested with Lys-C and trypsind.
Project description:To investigate the effect of RBPMS KO on mesoderm commitment we induced WT and RBPMS KO to mesoderm and isolated RNA at 0h (control),3h, 6h and 48h post induction.
Project description:Ribosome Occupancy Experiments: for these experiments Channel 1 is amplified RNA (aRNA) from mRNA unbound to any ribosomes. Channel 2 is aRNA from mRNA associated with ribosomes. Ribosome Density Experiments: for these experiments Channel 1 is amplified RNA (aRNA) from mRNA present in the light fractions of the polysome. Channel 2 is aRNA from mRNA associated with ribosomes deep in the polysome. Experiments are biological replicates of ribosome occupancy and density 12 hours post mock transfection or miR-124 transfection.
Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 M-NM-<g of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 M-NM-<g of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as M-bM-^IM-%5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).
Project description:Ribosome Occupancy Experiments: for these experiments Channel 1 is amplified RNA (aRNA) from mRNA unbound to any ribosomes. Channel 2 is aRNA from mRNA associated with ribosomes. Ribosome Density Experiments: for these experiments Channel 1 is amplified RNA (aRNA) from mRNA present in the light fractions of the polysome. Channel 2 is aRNA from mRNA associated with ribosomes deep in the polysome.
Project description:We reported loss of ARID1A promoted neurogenesis and inhibited cardiogenesis. Under microscopy, we observed that spontaneously differentiated cells were induced in ARID1A KO H9 hESCs cultured in mTesR medium. We did not know what cells types were. Here ATAC-seq were used to investigate chromatin accessibilities change in differentiated (day 4) WT H9 hESCs and ARID1A KO hESC cells.
Project description:We compared the ratio of polysome-associated mRNAs (>3 ribosomes/mRNA) normalized to total mRNA levels between WT and ppp-1 animals. We found a significant de-enrichment of 336 mRNAs and an enrichment for 72 mRNAs in ppp-1 polysome fractions.
Project description:Sucrose gradient ultracentrifugation followed by microarray analysis was used to identify translationally-regulated mRNAs (mRNAs associated with ribosomes) from JFH1-infected and uninfected Huh-7.5.1 cells. Huh7 cells were infected with HCV JFH-1 strain. Controls consisted in non-infected cells and infected cells treated with an anti-protease. 3 days after infection, cell lysates were subjected to sucrose-gradient centrifugation in order to isolate polysome-free RNAs from polysome-bound (translated) RNAs. The non-polysomal, polysomal as well as cytosolic (non fractionned) RNAs, from infected, non-infected, and infected-treated cells were subject sur microarray analysis.
