PRO-seq (Precision Run-On sequencing) to profile nascent RNA of H3.3 mutant (K9A K27A K79A) and control mESCs
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB61058 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR11178124.fastq.gz | Fastqsanger.gz | |||
| ERR11178125.fastq.gz | Fastqsanger.gz | |||
| ERR11178126.fastq.gz | Fastqsanger.gz | |||
| ERR11178127.fastq.gz | Fastqsanger.gz | |||
| ERR11178128.fastq.gz | Fastqsanger.gz |
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Project description:Mouse embryonic stem cells (i.e., mESCs; line ESC 129-B13) were genetically modified using CRISPR-Cas9 to mutate the H3f3b locus, in order to carry homozygous lysine-to-alanine substitution of residues K9 or K27. Two control mESC lines carrying knock-out of the H3f3a gene were used as background for the editing. To profile nascent RNA levels in H3.3 mutant and control mESCs, two rounds of PRO-seq experiment were performed. In the first round, three replicates each of H3.3K9A and control (i.e., Ctrl_9) mESCs were used. In the second round, two replicates each of H3.3K27A and control (i.e., Ctrl_27) mESCs were used. After permeabilization of the mESCs (and addition of 5% Drosophila S2 cells spike-in), a 2 biotin run-on reaction using biotin-11-UTP and biotin-11-CTP was carried out to label nascent transcripts. Biotinylated nascent RNA was then enriched as part of the library preparation process. Sequencing was performed on a NextSeq500 platform in single-end mode (75bp reads). The experiment allows for an assesment of the changes in the activity of cis-regulatory elements, upon removal of H3.3 K9 or K27 residues.
2024-09-04 | E-MTAB-12869 | biostudies-arrayexpress
Project description:Study to investigate the role of histone residues H3K4 and H3K36 for gene expression, histone localization and neuronal lineage specification by mutation of K4 and K36 in H3.3 to alanine. Histone variant H3.3 differs from the canonical H3.1/H3.2 by only 4 to 5 amino acids, which are necessary for nucleosome assembly independent of DNA replication, and is encoded by two gene copies. Complete loss of the two H3.3 genes (H3f3a and H3f3b) leads to embryonic lethality while single gene knockout yields viable mice. We used CRISPR-Cas9 to delete H3f3a and introduce homozygous point-mutations into H3f3b, thus ensuring that the entire pool of H3.3 protein carries the mutation of interest. We differentiated H3.3ctrl (H3f3a knock-out; H3f3b wild type), H3.3K4A mutant (H3f3a knock-out; H3f3b K4A) and H3.3K36A mutant (H3f3a knock-out; H3f3b K36A) ESCs into glutamatergic neurons. To assess the effect of the K4A mutation on Pol II activity, nascent RNA levels were mesaured by PRO-seq.
2020-01-28 | E-MTAB-8617 | biostudies-arrayexpress
Project description:PRO-seq in control and H3.3 K4A mutant mESCs
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Project description:We profiled the enrichment of the replicative H3.1 and non-replicative H3.3 histone variants using the SNAP-seq assay previously developed in the team (Gatto et al., 2022, Forest et al., 2023). We used KH2 mouse embryonic stem cells (mESCs) bearing doxycycline-inducible H3.1/H3.3-SNAP, which were expressed at low levels following 48h doxycycline treatment. To understand how the variants are distributed cell types of different potencies, we assayed them in pluripotent mESCs and following a 7-day differentiation into neural precursor cells (NPCs).
2025-04-24 | E-MTAB-14240 | 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:Precision nuclear run-on sequencing (PRO-Seq) data
| PRJNA717361 | ENA
Project description:Precision nuclear run-on (PRO) sequencing (PRO-seq) is a powerful technique for mapping transcriptomes with nucleotide resolution and measuring newly synthesized transcripts at both promoters and enhancer elements. The current PRO-seq protocol is time intensive, technically challenging, and requires a large amount of starting material. To overcome these limitations, we developed rapid PRO-seq (rPRO-seq), enabling efficient transcriptome mapping within a single day (∼12 h), increasing ligation efficiency, abolishing adapter dimers, and reducing sample loss and RNA degradation. Rapid PRO-seq allows for transcriptome mapping using 5,000 cells and is applicable to mouse hematopoietic progenitor cells (mHPCs) as well as mouse neurons. Using acute depletion of INTS11 in neuronal cells, we pinpoint a critical role for INTS11 as a regulator of genes in neurodevelopmental disorders. Taken together, rPRO-seq represents a significant advance in the field of nascent transcript analyses and will be a valuable tool for generating patient-specific genome-wide transcription profiles with minimal sample requirements.
2025-07-24 | GSE264740 | GEO
Project description:Mouse embryonic stem cells (i.e., mESCs; line ESC 129-B13) were genetically modified using CRISPR-Cas9 to mutate the H3f3b locus, in order to carry homozygous lysine-to-alanine substitution of residues K9, K27 or K79. Two control mESC lines carrying knock-out of the H3f3a gene were used as background for the editing. To compare the transcriptional profiles of the H3.3 mutant and control mESC lines, three independent replicates per condition were grown and RNA was extracted. After quality assessment of the RNA, messenger RNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs) and sequencing libraries were prepared using the NEBNext Ultra II RNA Library Preparation Kit for Illumina (New England BioLabs). Sequencing was performed on a NextSeq500 platform in single-end mode (75bp reads).
2024-03-09 | E-MTAB-12866 | biostudies-arrayexpress
Project description:Histone modifications are associated with distinct transcriptional states, but it is unclear whether they instruct gene expression. To investigate this, we mutated histone H3.3 K9 and K27 residues in mouse embryonic stem cells (mESCs). Here, we find that H3.3K9 is essential for controlling specific distal intergenic regions and for proper H3K27me3 deposition at promoters. The H3.3K9A mutation resulted in decreased H3K9me3 at regions encompassing endogenous retroviruses and induced a gain of H3K27ac and nascent transcription. These changes in the chromatin environment unleashed cryptic enhancers, resulting in the activation of distinctive transcriptional programs and culminating in protein expression normally restricted to specialized immune cell types. The H3.3K27A mutant disrupted deposition and spreading of the repressive H3K27me3 mark, particularly impacting bivalent genes with higher basal level of H3.3 at promoters. Therefore, H3.3K9 and K27 crucially orchestrate repressive chromatin states at cis-regulatory elements and bivalent promoters, respectively, and instruct proper transcription in mESCs.
2024-06-24 | MSV000095134 | MassIVE
Project description:mRNA-seq of H3.3 mutant (K9A K27A K79A) and control mESCs
| PRJEB61021 | ENA