Ontology highlight
ABSTRACT:
PROVIDER: PRJNA725062 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14322276_1.fastq.gz | Fastqsanger.gz | |||
| SRR14322276_2.fastq.gz | Fastqsanger.gz | |||
| SRR14322277_1.fastq.gz | Fastqsanger.gz | |||
| SRR14322277_2.fastq.gz | Fastqsanger.gz | |||
| SRR14322278_1.fastq.gz | Fastqsanger.gz |
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Project description:We report the updated method for CUT&Tag technology for high-throughput profiling of histone modifications or transcription factors in mammalian cells. By using using hand-made concanavalin A conjugated beads, we greatly improved handling and suspendability of cell-bound magentic beads and increased sensitivity for detecting peaks compared with conventional concanavalin A coated magnetic beads. This study provides a framework for the application of epigenome studies using mammalian cells.
2021-08-20 | GSE182254 | GEO
Project description:Purpose: Identification of glucocorticoid recepor binding sites on human kidney proximal tubular cells to better understand epigenetic alterations in diabetic kidney diseases Methods: CUT&RUN assay libraries for cultured cells or human kidneys were generated with the CUTANA kit (EpiCypher, 14-1048). 500,000 cells were mixed and incubated with Concanavalin A (ConA) conjugated paramagnetic beads. Antibodies to the glucocorticoid receptor were added to target samples and IgG was added to negative controls. The remaining steps were performed according to manufacturer’s instructions. Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, E7645S) with manufacturer’s instructions, including minor modifications indicated by CUTANA described above. CUT&RUN libraries were sequenced on a NovaSeq instrument (Illumina, 150 bp paired-end reads). For bulk ATAC-seq, 50,000 nuclei were transposed in 25 μL of ATAC-seq transposition mix [12.5 μL 2× Illumina Tagment DNA (TD) buffer; 10.5 μL nuclease-free water; 2.0 μL Tn5 transposase (Illumina/Nextera, FC-121-1030)] and incubated at 37°C for 1 h on a thermomixer. The transposed DNA was purified with QIAGEN MinElute kit (28004) and amplified with dual indexes.
2022-07-28 | GSE195443 | GEO
Project description:Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modelled this leukemic evolution through stepwise gene editing of GATA1s, SMC3+/- and MPLW515K providing 20 different trisomy or disomy 21 iPSC clones. Cut&Tag against GATA1 was performed on CD41+CD42+ megakaryocytes obtained after 18 days of differentiation of the IPSC clones. 500,000 CD41+CD42+ MK sorted cells were used to analyze GATA1 and GATA1s chromatin occupancy using the CUT&Tag-IT Assay Kit (Active Motif) according to the manufacturer recommendations. Briefly, cells were bound to Concanavalin A-Coated Beads and incubated with primary anti-GATA1 antibody in buffer with Protease Inhibitor Cocktail and 5% digitonine overnight at 4°C under rotation. The Guinea Pig anti-rabbit secondary antibody was incubated in Dig-Wash buffer for 1 hour at RT under rotation. After 3 washes, the CUT&Tag-IT™ Assembled pA-Tn5 Transposomes (1:100) were added for 1 hour at RT under rotation and tagmentation was performed during 1 hour at 37°C. DNA was purified and libraries were generated by PCR. The final libraries were purified, pooled together in equal concentrations and subjected to paired-end sequencing (100 cycles: 2x50) in Novaseq-6000 sequencer (Illumina) at Gustave Roussy.
2022-09-30 | E-MTAB-11032 | biostudies-arrayexpress
Project description:RNA m5C methylation profile of MCF10A and MDA486 by using MeRIP-Seq protocol Immunoprecipitation of Methylated mRNA at Cytosine (m5C) residues: Affinity purified of anti-methyl cytosine (m5C) polyclonal antibody 7ug (Zymo Research, Catalog#A3001-50) was conjugated with protein-A magnetic beads for 2 h at 4°C in end to end rotator. After that, conjugated beads were extensively washed with RNA immunoprecipitation (RIP) wash buffer to remove unbound antibody. Fragmented 25 ug polyA RNA (mRNA) was incubated with m5C conjugated beads for overnight at 4°C in in the rotating platform in RIP buffer. RIP was done using Megna RNA Immunoprecipitation kit (Millipore, Catalog#17-700). m5C mRNA-immune bead complex was treated with proteinase K buffer to release m5C mRNA from the conjugated antibody. To isolate m5C, mRNA was treated with phenol:chloroform:isoamyl and mixed with 400 ul of chloroform, which was centrifuged at 14000 rpm for 10 minutes to separate aqueous phase. The aqueous phase was ethanol precipitated at -80°C for overnight, to get m5C mRNA. This precipitated m5C mRNA pellet was washed twice with 70% ethanol and air dried. Finally, m5C mRNA pellet was dissolved in nuclease free Water. The m5C mRNA integrity and conentration was quantified by bioanalyzer (Agilent) and Qubit 2.0 flurometer (Invitrogen). The fragmented mRNA was used by following TruSeq RNA Sample Preparation Guide to develop RNA-Seq library for sequencing.
2014-02-20 | E-GEOD-53370 | biostudies-arrayexpress
Project description:We recently introduced CUT&Tag, an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei, and then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds DNA sequencing adapters (“tagmentation”) for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses exposure artifacts to ensure high-fidelity mapping of the antibody-targeted protein and improves signal-to-noise over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube from cells to amplified libraries, providing low-cost high-resolution genome-wide chromatin maps. By simplifying library preparation, CUT&Tag requires less than a day at the bench from live cells to sequencing-ready barcoded libraries. Because of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for ~$25 per sample, enabling routine genome-wide profiling of chromatin proteins and modifications that requires no special skills or equipment.
2020-07-15 | GSE145187 | GEO
Project description:We comprehensively benchmarked CUT&Tag for H3K27ac and H3K27me3 against published ChIP-seq profiles from ENCODE in K562 cells. Combining multiple new and published CUT&Tag datasets, there was an average recall of 54% known ENCODE peaks for both histone modifications. To optimize data analysis steps, we tested peak callers MACS2 and SEACR and identified optimal peak calling parameters. Considering both precision and recall of known ENCODE peaks, the peak callers were comparable in their performance, although peaks produced by MACS2 match ENCODE peak width distributions more closely. We found that reducing PCR cycles during library preparation lowered duplication rates at the expense of ENCODE peak recovery. Despite the moderate ENCODE peak recovery, peaks identified by CUT&Tag represent the strongest ENCODE peaks and show the same functional and biological enrichments as ChIP-seq peaks identified by ENCODE. Our workflow systematically evaluates the merits of methodological adjustments, providing a benchmarking framework for the experimental design and analysis of CUT&Tag studies, and will facilitate future efforts to apply CUT&Tag in human tissues and single cells.
2025-01-14 | GSE286492 | GEO
Project description:Purpose: this study is to analyze the chromatin landscape of H3R2mes in Plasmodium falciparum. Methods: In this study, H3R2me2s enrichment in the chromatin of wildtype parasite at schizont stage was analyzed by Cleavage Under Targets and Tagmentation (CUT&Tag) coupled with next generation sequencing (CUT&Tag-seq). Synchronized WT 3D7 at 40–46 hpi schizonts were harvested and fixed with 1% formaldehyde for 1 min at room temperature, followed by lysis with 0.06% saponin. The parasite pellets were suspended in 100 μl nuclear extraction buffer (20 mM HEPES–KOH pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine, 1x EDTA-free Protease Inhibitor cocktail). Ten μl of activated Concanavalin A-coated magnetic beads (EpiCypher # 21-1401) were added to each sample (~0.5 × 106 nuclei) and incubated for 10 min. The bead-bound nuclei were resuspended in 50 μl Antibody150 buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitor cocktail, 0.01% digitonin, 2 mM EDTA) containing 0.5 μg of rabbit anti-H3R2me2s (Epigentek #A-3705-050) as the primary antibody or rabbit IgG (Cell Signaling Technology #2729S) serving as a control. The primary antibody was then removed, and nuclei were then incubated with 0.5 μg of the secondary antibodies in 50 μl of Digitonin150 buffer (Antibody150, no EDTA) at room temperature for 30 min. The secondary antibodies were mouse anti-rabbit IgG (Invitrogen #31213). The nuclei were washed thrice with 200 μl Digitonin150 Buffer for 5 min to remove unbound antibodies and finally resuspended in 50 μl Digitonin300 Buffer (Digitonin150 but with 300 mM NaCl). Then, 2.5 μl of CUTANA pAG-Tn5 (EpiCypher #15-1017) were added to each reaction at room temperature for 1 h. The nuclei were washed thrice for 5 min in 200 μl Digitonin300 Buffer to remove the unbound pA-Tn5. Next, the nuclei were resuspended in 50 μl Tagmentation buffer (10 mM MgCl2 in Digitonin300) and incubated at 37 °C for 1 h. The beads were washed with 50 µl TAPS buffer (10 mM TAPS, pH 8.5, 0.2 mM EDTA), mixed with 5 µL SDS release buffer (10 mM TAPS pH 8.5; 0.1% SDS) to quench tagmentation, and then incubated for 1 hr at 58ºC to release tagmented chromatin into solution. Finally, 15 µl of 0.67% Triton-X was added to each reaction to quench SDS. PCR with appropriate barcoded primers was performed using the CUTANA High Fidelity 2x PCR Master Mix (EpiCypher #15-1018) according to the manufacturer's recommendations. Amplified DNA libraries were captured by incubating with 1.3 × Kapa pure beads (Roche #KK8001) according to the manufacturer's recommendations, and next-generation sequencing was performed on the NextSeq 550 platform. The reads of CUT&Tag-seq from three biological replicates were mapped to the P. falciparum genome (PlasmoDB Release 39.0) by using BWA (Li and Durbin 2009) after FastQC quality control. The peak calling was finished by MACS2 version 2.2.7.1 with parameters ‘callpeak -f BAMPE’ and a q-value cutoff of 0.05. Results: The three replicates of CUT&Tag-seq using nuclei from the schizont stage showed high reproducibility, with correlation coefficients of ~0.8. Peak calling using criteria of presence in at least two of three replicates with at least 50% overlap of the peaks identified 1629 H3R2me2s signals, distributed among the 14 chromosomes. Among these peaks, 57% (927) are localized at 5’ UTR regions of 791 genes, 35% (577) at coding regions of 470 genes, and 8% (125) at 3’ UTR regions of 115 genes. The peaks are ~17.44 bp wide and ~1265 bp from the ATG sites. Comparing the transcriptome data showed genes with H3R2me2s enrichment in the 5’ UTRs showed significantly higher expression than other genes in the genome, suggesting that H3R2me2s is associated with gene activation. In addition, H3R2me2s peaks were highly co-localized with the active mark H3K9Ac, H2A.Z, and H3K4me3, further implying H3R2me2s as a euchromatin mark. Gene ontology (GO) enrichment analysis revealed a multitude of cellular pathways, including merozoite invasion, transcription, translation, stress response, cell cycle, schizogony, cell adhesion, exit from RBC, vesicle transport, signal transduction, DNA repair, and telomere organization, which are potentially regulated by this euchromatic mark. Conclusions: Collectively, H3R2me2s is a active chromatin mark and regulates invasion and other important processes in human malaria parasite.
2023-06-28 | GSE214535 | GEO
Project description:We comprehensively benchmarked CUT&Tag for H3K27ac and H3K27me3 against published ChIP-seq profiles from ENCODE in K562 cells. Across a total of 30 new and 6 published CUT&Tag datasets we found that no experiment recovers more than 50% of known ENCODE peaks, regardless of the histone mark. We tested peak callers MACS2 and SEACR, identifying optimal peak calling parameters. Balancing both precision and recall of known ENCODE peaks, SEACR without retention of duplicates showed the best performance. We found that reducing PCR cycles during library preparation lowered duplication rates at the expense of ENCODE peak recovery. Despite the moderate ENCODE peak recovery, peaks identified by CUT&Tag represent the strongest ENCODE peaks and show the same functional and biological enrichments as ChIP-seq peaks identified by ENCODE. Our workflow systematically evaluates the merits of methodological adjustments and will facilitate future efforts to apply CUT&Tag in human tissues and single cells.
2022-03-31 | GSE199611 | GEO
Project description:We report the generation of single-cell RNAseq data obtained using Drop-seq from endothelial-enriched retinal cell suspension. Single-cell suspension were enriched using CD-31 antibody coated magnetic beads, and processed for encapsulation and library preparation as in Macosko et al (Cell 2015) based on the Drop-seq procedure.
2022-01-02 | GSE174400 | GEO
Project description:DNA and protein interactions are essential for cellular processes and have major implications in cancer biology. DNA binding proteins, particularly transcription factors (TF), are crucial in regulating gene expression and downstream protein functions. Studying the intricate DNA-protein interactions helps unravel specific functions of TFs that are involved in cancer pathogenesis, identify regulatory pathways, and ultimately uncover therapeutic targets. Currently, the lack of high-throughput screening techniques makes research in cancer associated-TFs challenging. Therefore, we have developed an innovative Iso-CUT&MAP (Isolated cell-based Cut Under Target and Mapping Apoptotic Protein-binding sequences). Here, K562 tumor cells were coencapsulated with NK92 and antibody-conjugated (NFkB/RelA) magnetic beads in a microfluidic droplet-based system. After NK92 immune cells killed the K562 target cells, we collected RelA-bound DNA from the dead cells and analyzed DNA-binding sites using ChIP-seq. The results reveal the RelA genome-wide association study (GWAS) of the target K562 tumor cells in response to immune cell killing. The genome-wide mapping matched the expected profile of NFkB, validating the ability of our Iso-CUT&MAP method to accurately identify NFkB binding in response to NK92 killing of K562. This novel technique holds promise for precision medicine in understanding apoptosis biology and the influence of immune cell or chemotherapy-induced tumor cell killings.
2024-07-19 | GSE237814 | GEO