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| Status |
Public on Mar 05, 2024 |
| Title |
KZ2658-GC4680002-Pro-seq_WT_Th1_Control_2 |
| Sample type |
SRA |
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| Source name |
CD4 Th1 cells
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| Organism |
Mus musculus |
| Characteristics |
cell lines: In vitro cultured mouse primery CD4+ T cells
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
PRO-seq (precision nuclear run-on sequencing) assays were performed as previously reported {Mahat, 2016 #10} with some modifications. Briefly, 500,000 cells were sorted from both Brg1-AID and control mice and permeabilized with ice-cold permeabilization buffer (1x106 cells/ml) on ice for 5 minutes. Then, the nuclear run-on reaction was carried out in the presence of biotin-dATP and unlabeled dCTP, dATP, dGTP, dUTP and sarkosyl. The nascent RNAs were extracted, fragmented, and purified by streptavidin pull-down. Next, 3' adapters were ligated directly to the purified nascent RNA. Following streptavidin purification, 5' ends were repaired using TAP and PNK and then ligated to 5' adapters. The adapter-containing RNA fragments are enriched by streptavidin pull-down, followed by reverse transcription
Reverse transcripted cDNA were PCR amplified with Illumina primers for 18 cycles, The fragments from 150bp to 700bp were isolated from E-gel and sequenced on Illumina NovaSeq.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
Pro-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 |
SAMN37006345 |
| SRA |
SRX21382776 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7713415_KZ2658-GC4680002-Pro-seq_WT_Th1_Control_2-mm10_0_0_mapq10_noDup.bed.uniq.graph.spikein.txt.gz |
4.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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