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Sample GSM3475866 Query DataSets for GSM3475866
Status Public on Mar 28, 2019
Title 3T3 fixed frozen 3000 cells IgG Ab-MNase, 400 mM salt wash buffer
Sample type SRA
 
Source name NIH/3T3 tet-on 3G cells
Organism Mus musculus
Characteristics cell line: 3T3
cell type: NIH/3T3 tet-on 3G cells
antibody-mnase: IgG Ab-MNase
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.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 3000
 
Description GB0460
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
 
Submission date Nov 16, 2018
Last update date Mar 30, 2019
Contact name Keji Zhao
E-mail(s) zhaok@nhlbi.nih.gov
Organization name national Institute of Health
Department National Heart, Lung, and Blood Institute
Street address Building 10 Room 7B06A
City Bethesda
State/province MD
ZIP/Postal code 20814
Country USA
 
Platform ID GPL21493
Series (1)
GSE105012 Single cell chromatin immunocleavage sequencing (scChIC-Seq) to profile histone modification
Relations
BioSample SAMN10434875
SRA SRX5016883

Supplementary file Size Download File type/resource
GSM3475866_KZ1235-7_0_0_mapq10_noDup.bed.gz 2.3 Mb (ftp)(http) BED
GSM3475866_KZ1235-7_mapq10_noDup_RPBM.bedgraph.gz 1.6 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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