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| Status |
Public on Mar 05, 2024 |
| Title |
Mice fibroblast-H3K27ac-CHIC-WT-with TSA-1#-New cells |
| Sample type |
SRA |
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| Source name |
Fibroblasts
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| Organism |
Mus musculus |
| Characteristics |
cell lines: Fibroblasts
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
cells for ChIC-seq were fixed for 10 minutes with 1% formaldehyde and permeabilized with RIPA buffer (10 mM Tris-Cl, 150 mM NaCl, 0.1% SDS, 0.1% NaDOC, 1% Triton X-100) for 10 minutes at RT. Antibodies (anti-H3K4me3 (17-614, Millipor and protein A-MNase fusion protein were mixed well and incubated at 4°C for 30 minutes. Next, the antibody-pA-Mnase complex was added to the permeabilized cells and incubated at 4°C for 1 hour. Then, the cells were washed with 600 μl RIPA buffer four times and rinsed once with 200 μl rinsing buffer (10 mM Tris-Cl and 10 mM NaCl, 0.1% Triton X-100). The MNase digestion was initiated by re-suspending rinsed cells in 40 μl reaction solution buffer (10 mM Tris-Cl, 10 mM NaCl, 0.1% Triton X-100, 2 mM CaCl2) and incubated at 37°C for 3 minutes. The reaction was stopped by adding 80 μl stop buffer (20 mM Tris-Cl, 10 mM EGTA, 20 mM NaCl, 0.2% SDS) and 1 μl proteinase K, then incubated at 65°C for overnight. DNAs were purified by using MinElute reaction cleanup kit.
DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3’ to 5’ exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. The ligation reaction was performed by adding 2 μM Illumina's adapters to each sample and incubated at RT one hour. After adapter ligation DNA was PCR amplified with Illumina primers for 16 cycles and library fragments from 180bp to 300bp were isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on Hi-seq3000 following the manufacturer's protocols.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
ChIC-seq
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| Data processing |
Basecalls performed
Reads from Pro-seq, DNase-seq and ChIC-seq were mapped to mouse genome (mm10) using Bowtie2. Reads with MAPQ ≤ 10 or redundant reads that mapped to the same location and orientation were removed. Reads from RNA-seq data were mapped to mouse genome (mm10) using Bowtie.
Paired-end sequencing reads for MNase-seq were mapped to mouse genome (mm10) using Bowtie2. Discordantly aligned reads, reads with mapping quality (MAPQ) ≤10, and redundant reads were removed from further analysis. We selected the fragments with range of 140-180bp as canonical nucleosomes for further analysis. The midpoints of nucleosomes were defined as their positions. We used center-weighted occupancy score to plot aggregated nucleosome profiles at single base resolution. Specifically, we defined the nucleosome center positioning (NCP) score S_k at position k, as the normalized nucleosome count with center at position k. We defined center-weighted occupancy score at position k as∑_(j=-73)^(j=73)▒S_(k+j) w_j, where w_j is the Gaussian weight equal to e^(-(j/20)^2/2).
Assembly: mm10
Supplementary files format and content: bedgraph files for Pro-seq, DNase-seq and ChIC-seq::
Supplementary files format and content: bedgraph files for RNA-seq:: normlized by the library size and using a window size of 200bp
Supplementary files format and content: WT_4000_3lanes.bed.140180.chr10_11.begraph_kernal20bp.txt.gz: normlized by the library size and using a window size of 20bp
Supplementary files format and content: KO_4000_3lanes.bed.140180.chr10_11.begraph_kernal20bp.txt.gz: combine both WT replicates
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| Submission date |
Aug 16, 2023 |
| Last update date |
Mar 05, 2024 |
| 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) |
| GSE156569 |
BRG1 contributes to the global chromatin accessibility revealed by its acute depletion |
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| Relations |
| BioSample |
SAMN37006338 |
| SRA |
SRX21382811 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7713408_GC3728011_H3K27ac_CHIC_WT_with_TSA_1_mm10_0_0_mapq10_noDup.bed.uniq.txt.graph.spikein.txt.gz |
47.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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