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SRX5016995: GSM3475978: human white blood cells single cells H3K4me3 Ab-Mnase-100; Homo sapiens; OTHER
1 ILLUMINA (Illumina HiSeq 3000) run: 9.6M spots, 477.5M bases, 137.9Mb downloads

Submitted by: NCBI (GEO)
Study: Single cell chromatin immunocleavage sequencing (scChIC-Seq) to profile histone modification
show Abstracthide Abstract
We report a single-cell chromatin immunocleavage sequencing (scChIC-seq) methodology for analyzing histone modifications, which involves targeting of the micrococcal nuclease (MNase) by tethering it to an antibody and selective PCR amplification of cleaved target sites. We show that the protocol reliably detects the H3K4me3 and H3K27me3 target sites in single human white blood cells (WBC), resulting data for successful identification of unique blood cell types based on clustering analysis. Overall design: The technique, scChIC-seq, was applied to profile H3K4me3 and H3K27me3 using small number of cells and single cells. By comparing with the Bulk ChIP-seq signal, scChIC-seq was shown to have high specificity. scChIC-seq was applied to profile H3K4me3 using single human white blood cells. The highly co-methylated and highly variable peaks were shown to be related to underlying cellular populations.
Sample: human white blood cells single cells H3K4me3 Ab-Mnase-100
SAMN10434724 • SRS4050126 • All experiments • All runs
Organism: Homo sapiens
Library:
Instrument: Illumina HiSeq 3000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: The DNAs were end-repaired using the End-It™ DNA End-Repair Kit (Lucigen, Radnor, PA, USA). The reaction was done in 10 µL reaction volume according to the manufacturer's instruction. The reaction was terminated by adding 115 µL 10 mM Tris-Cl buffer (pH7.5) containing 1 mM EDTA, and the DNAs were extracted by the phenol-chloroform extraction, followed by the ethanol precipitation as described above. The precipitate is re-suspended in 5 µL Qiagen Buffer EB (10 mM Tris-Cl buffer (pH 8.5)). The addition of 3' A overhangs to the end-repaired DNA fragments was done using the Klenow fragment (3'→5' exo-) (New England BioLabs, Ipswich, MA, USA) and 1 mM deoxyadenosine triphosphate in the reaction buffer. The DNA fragments were incubated at 37°C for 20 min and the enzymes were inactivated at 75°C for 20 min. The Illumina Adaptor Oligo Mix (Illumina, San Diego, CA, USA) was ligated to the 3' A overhangs of the DNA fragments using the T4 DNA ligase (New England BioLabs) by incubating at room temperature for 3 hr. The DNA fragment with the adaptors bound was PCR amplified using the Phusion High-Fidelity PCR Master Mix (New England BioLabs) and the 125 nM PE PCR Primer 1.0 (Illumina). The PCR was done under the following condition: 98°C for 10 min, followed by a cycle of 30 sec at 68°C and 30 sec at 72°C (repeat 18 cycles in the case of 3,000 cells or 25 cycles in the case of a single cell). The 2% [w/v] agarose gel electrophoresis was done to isolate the 140-350 bp fragments and purify using the QIAquick Gel Extraction kit (Qiagen). The concentration of the purified DNAs was measured using Qubit dsDNA HS kit (Thermo Fisher Scientific). The paired-end sequencings were run using the Illumina MiSeq and HiSeq2000.
Experiment attributes:
GEO Accession: GSM3475978
Links:
Runs: 1 run, 9.6M spots, 477.5M bases, 137.9Mb
Run# of Spots# of BasesSizePublished
SRR81976189,550,251477.5M137.9Mb2019-03-28

ID:
6770467

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