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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 28, 2019 |
| Title |
3T3 fixed frozen 3000 cells IgG Ab + PA-MNase, 200 mM salt wash buffer |
| Sample type |
SRA |
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| Source name |
NIH/3T3 tet-on 3G cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: 3T3
cell type: NIH/3T3 tet-on 3G cells
antibody-mnase: IgG Ab + PA-MNase
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| Extracted molecule |
genomic DNA |
| Extraction 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.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
GB0468
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| Data processing |
Basecalls performed using RTA 1.7 or Illumina Casava1.7
scChIC-seq reads were mapped to the mouse genome (mm9) using Bowtie2 (bowtie2 -p 18 -q -5 0 -3 0). The reads with MAPQ <=10 or redundant reads were removed for further analysis in each library.
The enriched regions were identified using SICER. For H3K4me3, Gap size = 200, win size = 200 fragment = 150. For H3K27me3, Gap size = 600, win size = 200 fragment = 150,
Genome_build: mm9
Supplementary_files_format_and_content: scoreisland: plan text file. Each line is a peak region. The four columns are chrom, start position, end position, score.
Supplementary_files_format_and_content: bed: Bed 6 file. Each line is a read. The six columns are chrom, start position, end position, end position-start position, mapq, strand
Supplementary_files_format_and_content: bedgraph: bed 4 file. Each line is a bin, with bin size = 200bp. The four columns are chrom, start position, end position,read density.
Library strategy: scChIC-seq
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| Submission date |
Nov 16, 2018 |
| Last update date |
Mar 30, 2019 |
| Contact name |
Keji Zhao |
| E-mail(s) |
zhaok@nhlbi.nih.gov
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| Organization name |
national Institute of Health
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| Department |
National Heart, Lung, and Blood Institute
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| Street address |
Building 10 Room 7B06A
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20814 |
| Country |
USA |
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| Platform ID |
GPL21493 |
| Series (1) |
| GSE105012 |
Single cell chromatin immunocleavage sequencing (scChIC-Seq) to profile histone modification |
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| Relations |
| BioSample |
SAMN10434870 |
| SRA |
SRX5016887 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3475870_KZ1241-5_0_0_mapq10_noDup.bed.gz |
381.5 Kb |
(ftp)(http) |
BED |
| GSM3475870_KZ1241-5_mapq10_noDup_RPBM.bedgraph.gz |
269.0 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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