ABSTRACT: SILAC quantitative proteomics was used to compare the whole cell proteomes of the differentially SILAC-labelled wild-type and acentrosomal (stil KO) DT40 cell lines.
Project description:SILAC quantitative affinity purification experiment was performed to compare the protein composition of pericentriolar satellites in the wild-type and acentrosomal (stil KO) DT40 cell lines.
Project description:Label-free quantitative affinity purification experiments were performed to identify the protein composition of pericentriolar satellites in the wild-type and two acentrosomal (stil KO; cep152 KO) DT40 cell lines.
Project description:Transcription profiling of chicken Pax5 deficient DT40 B cell line to investigate the targets of Pax5 which is required for B-cell differentiation Used in cross-species comparison to investigate evolutionarily conserved regulatory circuits in B cell development Pax5 deficient DT40 B cells (3 biological replicates) were compared to DT40 wild-type cells (3 biological replicates).
Project description:In chicken DT40 cells, there are six linker histone H1 variants and 12 of coding genes. We have previously reported of 11 out of 12 H1 knock out DT40 cells (Takami et al., Genes to Cell 1997 [PMID:9491804]) but complete H1 null DT40 cells could not established, so far. We identified one of the H1 variant, H1R was involved in genomic instabilities (Hashimoto et al., DNA repair (2007) [17613284]), so we re-introduced floxed H1R-eGFP and mer-cre-mer into 11 out of 12 H1 knock out DT40 cells. Then we targeted last enedogenous H1, we successfully established conditional H1 KO cells (K11). Next we treated with tamoxifen to loop out floxed H1R-eGFP, and cloning H1 completely null cells (K11-5, and K11-7). We analysis those gene expression pattern in wild-type, K11, and K11-5 cells Experiment Overall Design: Apoptosis is induced in H1 null cells, so we inhibit apoptosis with pan-caspase inhibitor, Z-VAD-FMK and extract RNAs.
Project description:Initiation and completion of DNA replication has a major impact on chromatin, but how core chromatin processes are cell cycle regulated in somatic cells remains poorly understood. To address this question we conducted a quantitative proteomics study that took advantage of an analogue sensitive mutation in Cdk1 (cdk1as), which allows for rapid and highly specific inactivation of Cdk1 by the bulky ATP analogue 1NMPP1. Neither Cdk1 nor Cdk2 are required for S-phase progression, while inactivation of Cdk1 in Cdk2 knock-out cells causes a complete block of DNA replication initiation and S-phase progression. Here we compared chromatin fractions obtained from cdk1as and cdk1as/cdk2-/- cells two hours after Cdk1 inactivation. Statistical analysis of these four independent experiments (including 2 label swaps) identified 135 proteins that showed a significant change in SILAC ratio among a total of 2402 proteins quantified in at least three experiments. This approach led us to discover a novel functional interplay between interphase Cdks and the chromatin association of a variety of novel candidate proteins such as Cdk1 itself, the APC/C regulatory subunit cdc20, the PP4 regulatory subunit Smek2, the helicase FUBP1 and the PHD domain-containing zinc finger protein PHF6.
Project description:RNA-seq was performed for transcriptional analysis of wild-type DT40 cells (Gallus gallus, B-cell line) and a H3.3 knockout line (h3.3a-/-, h3.3b-/-). H3.3 is a H3 histone variant encoded on two genes (H3.3A and H3.3B) in chickens.
Project description:Purpose: to identify how condensin I removal affects gene expression globally. Methods: Chicken DT40 CAP-H KO cells were treated with or without dox for 36 h. Total RNA samples were extracted and subjected to sequencing using an Illumina Hiseq2000 platform. Library preparation and Illumina sequencing were performed by Macrogen (South Korea). The sequence tags were spliced-mapped onto the chicken genome galGal4 using Tophat and Bowtie2 following quality test using FASTQC. Differential expression of genes was analyzed using the Bioconductor v2.3 package edgeR v3.2.3. Tag enrichment in NCBI RefSeq genes was calculated between dox-treated and untreated cells using edgeR exact test with tag-wise dispersion estimation. Results: We identified a total of 3798 genes to be differentially expressed in G1 condensin I-depleted chicken DT40 cells: 2495 down regulated and 1303 up regulated. Conclusions: Removal of CAP-H leads to significant misregulation of gene expression, suggesting a key role for condensin I in transcription during interphase. Total RNA profiles of CAP-H KO cells with or without Dox were generated by sequencing using Illumina Hiseq2000 platform.
Project description:Global gene expression profiling of the avian B-lymphoma DT40 cell line was used as a model to differentiate among Btk KO and Btk KO cells reconstituted with human Btk. Differences in the gene expression pattern showed statistically significant changes between parental DT40 and all the Btk KO cell populations irrespective of whether they are reconstituted or not. These results imply that in the process of generating a knockout cell line, subclones are selected, which have multiple changes in their gene expression pattern (p<0.01). Experiment Overall Design: DT40 cells along with the mutants were grown at various time points in different batches of fetal calf serum with or without any stimulation. All experiments were repeated ten times and then polled together as one sample. In total 28 samples were used RNA extraction and hybridization on Affymetrix microarrays.
Project description:A single replicate of exponentially growing DT40 CL18 chicken B lymphoma cells were harvested and extracted RNA was subjected to Illumina GAIIx paired-end sequencing to determine global gene expression. Single replicate RNA-seq expression analysis of DT40 cells.