Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Quantitative multiplexed ChIP-Seq (MINUTE-ChIP) from formaldehyde-fixed material

ABSTRACT: In crosslinked MINUTE-ChIP, formaldehyde-fixed chromatin is fragmented using sonication, blunted and ligated to double-stranded DNA adaptors that include a T7 RNA polymerase promoter and a sample barcode sequence. Finally, samples are combined and subsequent ChIP reactions are performed with the pooled samples. ChIP material is prepared into an Illumina-compatible library using linear amplification by virtue of T7 RNA polymerase, reverse transcription and a low-cycle library PCR amplification. Here, we demonstrate a MINUTE-ChIP for Nanog from formaldehyde-fixed cells using fragmentation by sonication.

ORGANISM(S): Mus musculus

PROVIDER: GSE244908 | GEO | 2024/02/11

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Quantitative multiplexed ChIP-Seq (MINUTE-ChIP) Calibration Experiment

Project description:In MINUTE-ChIP, native chromatin is fragmented using Micrococcal nuclease, and subsequently blunted and ligated to double-stranded DNA adaptors that include a T7 RNA polymerase promoter and a sample barcode sequence. Finally, samples are combined and subsequent ChIP reactions are performed with the pooled samples. ChIP material is prepared into an Illumina-compatible library using linear amplification by virtue of T7 RNA polymerase, reverse transcription and a low-cycle library PCR amplification. Native MINUTE-ChIP is based on Mint-ChIP, developed by the Bernstein lab (van Galen et al., 2016; PMID: 26687680). We have introduce unique molecule (UMI) counting and paired-end mapping of the chromatin fragments to this method, which we then termed MINUTE-ChIP for multiplexed indexed unique molecule T7 amplification end-to-end sequencing. Here, we generate a standard curve for H3K27me3 and demonstrate that MINUTE-ChIP has a large linear dynamic range, thus MINUTE-ChIP quantitation is proportional to real quantities.

2019-06-21 | GSE133056 | GEO

One-pot universal NicE-seq: All enzymatic downstream processing of 4% formaldehyde crosslinked cells for chromatin accessibility genomics

Project description:Accessible chromatin landscape allows remodeling of transcription factors and enhancers during development and molecular subtypes of cancer in patient samples. One-tube universal NicE-seq (OuNicE-seq) is an improved accessible chromatin profiling method that negate DNA purification and incorporate sonication free enzymatic fragmentation before library preparation and is suited to a variety of mammalian cells. OuNicE-seq is versatile, capable of profiling 4% formaldehyde fixed chromatin in 25 fixed cells and opens the possibility for automation. Accessible chromatin profile is more efficient on formaldehyde fixed cells using OuNicE-seq compare to Tn5 transposon mediated methods, demonstrating its versatility.

2021-12-01 | GSE175651 | GEO

Tead1 regulation of β-cells II

Project description:Purpose: The goal of this study is to determine the regulatory role of Tead1 in β-cells by analyzing the Tead1 cistrome and open chromatin in β-cells Methods: Isolated islets from WT C57bl6 mice of 12 weeks of age were flash frozen. For Chip-seq they were fixed with formaldehyde and then after sonication, IP was performed with Anti-Tead1 antibody or the IgG isotype control. For ATAC-seq 100,000 of the frozen nuclei were tagmented. After DNA extraction, library construction, sequencing was performed using Illumina Hi seq2500. Conclusions: Our study represents the first detailed analysis of the Tead1 cistrome in beta cells.

2022-12-14 | GSE157513 | GEO

ChIP-Seq experiment to analyse Zip1 localisation in the Ctf19c mutants during meiotic prophase.

Project description:The goal of this experiment is to confirm whether Ctf19c mutants affect Zip1 localisation during meiotic prophase. To do so, diploid S. cerevisiae SK1 cells were constructed containing ndt80-d to arrest cells in prophase. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication) Diploid S. cerevisiae SK1 cells were constructed containing ndt80-d to arrest cells in prophase. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication)

2015-12-15 | E-GEOD-70029 | biostudies-arrayexpress

Rec8 ChIP-Seq experiment to analyse cohesin localisation in the Ctf19c mutants during meiotic prophase.

Project description:Ctf19c mutants were shown to specifically impair cohesin localisation at the pericentromere during mitosis. The goal of this experiment is to confirm whether this mutant has the same effect on cohesin localisation during meiotic prophase. To do so, diploid S. cerevisiae SK1 cells were constructed containing Rec8-3HA in an ndt80-d background. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication) Diploid S. cerevisiae SK1 cells were constructed containing Rec8-3HA in an ndt80-d background. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication)

2015-12-15 | E-GEOD-70030 | biostudies-arrayexpress

Quantitative multiplexed ChIP-Seq (MINUTE-ChIP) Standard Curve

Project description:Quantitative multiplexed ChIP-Seq (MINUTE-ChIP) Standard Curve

| PRJNA549856 | ENA

Maximizing linear amplification of nucleic acids with optimized T7 promoters

Project description:Linear amplification of RNA by T7 bacteriophage polymerase is widely used in molecular biology. We performed 5’RACE-Seq to identify T7 promoter variants with enhanced transcriptional activity that generate up to five-fold higher RNA output in large scale synthesis reactions. In single-cell RNA-Sequencing, optimized T7 promoters facilitate library preparation, and substantially increase library complexity and the number of expressed genes detected per cell, highlighting a particular value for bioanalytical applications

2020-12-24 | E-MTAB-8508 | biostudies-arrayexpress

ChIP-Seq experiment to analyse Zip1 localisation in the Ctf19c mutants during meiotic prophase.

2015-12-15 | GSE70029 | GEO

ChIP-seq of RNA polymerase II using pan-pol II and S2-phosphorylated C-terminal domain antibodies in mouse embryonic stem cells

Project description:Our proteomics data (PXD030253) indicate RNA polymerase II is co-localized with Aldh18a1. To confirm this observation using an orthogonal method we performed ChIP-seq experiments to identify the binding patterns of RNA polymerase II in general, and RNA polymerase II with S2-phosphorylated CTD. We also performed Cut&Run using Aldh18a1 antibody which is submitted separately. To do a ChIP-seq, mES cells (46C) were treated with Accutase to be detached. The cells were fixed by 1.5% formaldehyde in PBS for 15min. Chromatin was fragmented by sonication and sheared to < 500 bp fragments. The cell debris were removed by spinning. The sheared chromatin was treated with 10 ug antibodies (pan-Pol II Bethyl A304-405A, or S2P-Pol II Bethyl A304-407A) overnight at 4 °C. Dynabeads protein A beads (40ul) were added to the samples and the samples were rotated 2-3 hours to perform the immunoprecipitation. The beads were washed 5 times. Chromatin was eluted in a denaturing solution, and proteins were digested using proteinase K. DNA was purified by SPRI beads. The ChIP libraries were prepared using NEBNext Ultra II DNA library prep according to the manufacturer manual. The libraries were sequenced using 150nt reads on paired-end mode by HiSeq4000. Reads were trimmed, aligned to the mouse genome (mm10) using Bowtie2, and duplicated reads were removed with samtools. The ChIP quality was evaluated by cross-correlation using the “SPP'' tool as suggested by ENCODE ChIP-seq guidelines. Peak calling was performed using MACS2.

2022-06-01 | E-MTAB-11510 | biostudies-arrayexpress

High throughput single cell epigenomic profiling by targeted insertion of promoters (TIP-seq)

Project description:Chromatin profiling in single cells has been extremely challenging and almost exclusively limited to histone proteins. In cases where single cell methods have shown promise, many require highly specialized equipment or cell type specific protocols and are relatively low throughput. Here, we combine the advantages of tagmentation, linear amplification and combinatorial indexing to produce a high throughput single cell DNA binding site mapping method that is simple, inexpensive and capable of multiplexing several independent samples per experiment. Targeted Insertion of Promoters (TIP-seq) uses Tn5 fused to protein A to insert a T7 RNA polymerase promoter adjacent to a chromatin protein of interest. Linear amplification of flanking DNA with T7 polymerase prior to sequencing library preparation provides ~10-fold higher unique reads per single cell compared to other methods. We apply TIP-seq to map histone modifications, RNA Polymerase II (RNAPII) and transcription factor CTCF binding sites in single human and mouse cells.

2021-11-10 | GSE188512 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data