Mapping of nascent RNA in MLL2 F/F and FC/FC MEF cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE73126 | GEO | 2016/04/13
SECONDARY ACCESSION(S): PRJNA296117
REPOSITORIES: GEO
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Project description:Global Run-On has been performed in MLL2 F/F and FC/FC MEF cells. The aim of the experiment is to test how the lack of MLL2 affects nascent RNA transcription. Four samples were analyzed - 2 independent replicates. The experiments were performed using immortalized mouse embryonic fibroblasts (MEF) in which both MLL2 alleles were targeted by the loxp system (F/F cells). Tamoxifen treatment of the F/F cells for 24 hours results in the excision of both MLL2 alleles (FC/FC cells).
2016-04-13 | E-GEOD-73126 | biostudies-arrayexpress
Project description:We investigated the RNAPII and γH2AX occupancy genome wide by ChIP-Seq in MLL2 F/F and FC/FC80 MEF cells. We found that a week after MLL2 excision (FC/FC cells), a group of genes present higher levels of γH2AX and RNAPII near the TSS, as compared to the control (F/F cells). H3K4Me1, H3K4M2 and H3K4Me3 levels near the TSS were also studied.
2016-04-13 | GSE73124 | GEO
Project description:We investigated the RNAPII and γH2AX occupancy genome wide by ChIP-Seq in MLL2 F/F and FC/FC80 MEF cells. We found that a week after MLL2 excision (FC/FC cells), a group of genes present higher levels of γH2AX and RNAPII near the TSS, as compared to the control (F/F cells). H3K4Me1, H3K4M2 and H3K4Me3 levels near the TSS were also studied. There is a total of 52 samples. 3 independent replicates for each experiment were performed. H3, H2AX and IgG ChIPs were used for normalisation or as controls.The experiments were performed using immortalised mouse embryonic fibroblasts (MEF) in which both MLL2 alleles were targeted by the loxp system (F/F cells). Tamoxifen treatment of the F/F cells for 24 hours results in the excision of both MLL2 alleles (FC/FC cells).
2016-04-13 | E-GEOD-73124 | biostudies-arrayexpress
Project description:Mapping of nascent RNA in MLL2 F/F and FC/FC MEF cells
| PRJNA296117 | ENA
Project description:We used human or mouse MLL2-impaired cells (including a human DSET MLL2 cell line,) as well as their parental controls, and measured mutations in histones and a few other genes that were affected in our gH2AX ChIP-Seq experiments. We found that cells lacking or having impaired MLL2 were presenting statistically signifficant, higher levels of mutations at the tested genes as compared to their parental controls. The experiments were performed using human and mouse cells. For the human cells, the parental HCT116 cells were used as control. Two different MLL2 KO clones (KO19 and KO34) and a DSET MLL2 cell line were employed. For the mouse cells, the F/F cells were used, in which both MLL2 alleles are targeted by loxp. Tamoxifen treatment for 24 hours results in the excision of both MLL2 alleles (FC/FC cells). F/F cells were treated with tamoxifen (FC/FC) or vehicle (DMSO, F/F) for 24 hours. Half of the cells were then harvested and used as controls (T0), while the other half were cultured for another 30 days (T30). Mutations after 30 days (T30) of tamoxifen/vehicle treatment were measured compared to the T0 controls. In all cases, two different pools of primers were employed. All histone genes available to target with high coverage were studied, in either human or mouse cells. For the human cells, 32 histone (HIST1H1A HIST1H1B HIST1H1C HIST1H1D HIST1H1E HIST1H1T HIST1H2AC HIST1H2AD HIST1H2AG HIST1H2AH HIST1H2AI HIST1H2AJ HIST1H2AK HIST1H2BF HIST1H2BG HIST1H2BH HIST1H2BI HIST1H2BJ HIST1H2BK HIST1H2BN HIST1H3B HIST1H3C HIST1H3D HIST1H3E HIST1H3F HIST1H3G HIST1H3H HIST1H3I HIST1H4B HIST1H4F HIST1H4J HIST2H2AC) and 6 other genes (BTG2 SGK1 MDM2 CDKN1A EGR1 RPLP0) were targeted. For the mouse cells, 21 histone (HIST1H1A HIST1H1B HIST1H1C HIST1H1D HIST1H1E HIST1H1T HIST1H2AK HIST1H2BQ HIST1H3B HIST1H3C HIST1H3F HIST1H4F HIST1H4J HIST1H4N HIST1H4N HIST2H2AA1 HIST2H2AA1 HIST2H2AA2 HIST2H2AA2 HIST2H2AC HIST2H2BB) and 6 other genes (BTG2 SGK1 MDM2 CDKN1A EGR1 RPLP0) were targeted. Processed data to be submitted to appropriate variation archive.
2016-04-13 | E-GEOD-73128 | biostudies-arrayexpress
Project description:CUT&RUN-seq of H3K4me3, H3K27me3 and H3K27ac in mouse ESCs carrying a homozygous point mutation in the catalytic domain of mll2[Y2602A]. CUT&RUN was performed according to Skene & Henikoff, 2017 and purified DNA was using for library preparation with NEBNext DNA Ultra II kit (NEB E7645S; input: 5 ng of DNA). Libraries were multiplexed and sequenced on a NextSeq500 (Paired-End; read length 40). Each sample is present in 3 biological replicates.
2023-11-15 | E-MTAB-13466 | biostudies-arrayexpress
Project description:Global Run-On has been performed on WT or KD for RECQL5 cells after release from DRB. When RECQL5 is knocked-down the transcriptional wave front is more advanced, suggesting that transcription is faster. Constitutive knock-down cell lines expressing or not endogenous levels of shRNA resistant RECQL5 under a Doxycycline inducible promoter were treated with high doses of DRB to block transcription. Upon release into fresh medium we were able to follow how much and how fast the RNA Pol II progresses through genes by mapping nascent RNA by Run-On. The experiment was performed in two cell line clones.
2014-06-09 | E-GEOD-49133 | biostudies-arrayexpress
Project description:RNA sequences are expected to be identical to their corresponding DNA sequences. Advances in technologies have enabled deep sequencing of nucleic acids that uncovered exceptions to the one-to-one relationship between DNA and RNA sequences. Previously in human cells, post-transcriptional RNA editing was the only known mechanism that changes RNA sequences from the underlying DNA sequences. Here, we sequenced nascent RNA and found all 12 types of RNA-DNA differences. Using various experimental analyses, we validated this finding. Our results showed that sequences of nascent RNAs within 40 nucleotides of the exit channel of RNA polymerase II already differ from the corresponding DNA sequences. These RNA-DNA differences are mediated by RNA processing steps closely coupled with transcription and not by known deaminase-mediated RNA editing mechanisms nor during NTP incorporation by Pol II. This finding identifies sequence substitution as part of co-transcriptional RNA processing. We sequenced nascent RNA using global run-on sequencing, GRO-seq from human B-cells from two individuals and a variant of the GRO-seq procedure, known as precision run-on sequencing, PRO-seq. The RNAs are prepared after a short run-on assay performed with isolated nuclei in the presence of Br-UTP. The isolated RNAs are base hydrolyzed to ~100 nucleotides and affinity purified with anti-BrU beads three times at each successive step of preparing the RNAs for orientation-specific sequencing using Illumina technology. The 5M-bM-^@M-^Y ~half of each sequence represents nascent RNA made in the cell and the 3M-bM-^@M-^Y ~half represents sequences made in vitro during the run-on reaction. The precision variation, PRO-seq, incorporates one or at most a few biotin-labeled nucleoside triphosphates during the run-on, and sequencing from the 3M-bM-^@M-^Y end of this affinity purified, nascent RNA maps the cellular location of engaged polymerases with near single nucleotide precision. We obtained ~ 100 million 100-nucleotide uniquely mapped GRO-seq reads from B-cells of two individuals. For one subject, we also carried out pGRO-seq and obtained 60 million uniquely mapped reads. In addition, we sequenced ~135 million uniquely mapped RNA-seq reads, and the corresponding DNA of the two individuals to 30X and 60X coverage. Additionally, we isolated and sequenced nascent RNA with an alternate method described by Wuarin and Schibler (1994) in order to compare chromatin-bound RNA to the very nascent RNA from PRO-seq. We obtained ~190 million uniquely mapped reads from chormatin-bound RNA-seq.
2014-02-21 | E-GEOD-39878 | biostudies-arrayexpress
Project description:Geitlerinema sp. FC II strain:FC II Genome sequencing and assembly
| PRJNA399071 | ENA
Project description:We combined the nuclear run-on (NRO) assay which labels and captures nascent transcripts with high throughput DNA sequencing to examine transcriptional activity in Saccharomyces cerevisiae. Examination of nascent transcripts and steady-state transcripts in exponentially growing and heat-shock treated yeast.
2011-11-01 | E-GEOD-33136 | biostudies-arrayexpress