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| Status |
Public on May 30, 2024 |
| Title |
KO_ChIP, rep 1, ChIPseq |
| Sample type |
SRA |
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| Source name |
Spleen
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| Organism |
Mus musculus |
| Characteristics |
tissue: Spleen
antibody: OCT2 ChIP
cell type: NK cells
genotype: KO
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cultured NK cells from Reg-1fl/fl and Reg-1ΔNK were cross-linked with formaldehyde (final concentration; 1%) followed by quenching with glycine. Wash the cells with PBS and resuspend the cell pellet with cell lysis buffer for ChIP and incubate for 10 min on ice. Pellet the cell nuclei by centrifugation, then resuspend the nuclear pellet in nuclei lysis buffer for ChIP followed by chromatin fragmentation with Bioruptor (Sonicbio Co., Ltd.).
Purified ChIP-DNA was applied for library preparation by DNA SMART ChIP-Seq Kit (TaKaRa) following the manufacturer’s instructions. Briefly, each 1 ng of purified ChIP-DNA demonstrated denaturation, dephosphorylation of 3’-Ends, and T-Tailing, then dsDNA was produced by Poly(dA) primer annealing and template-switching reaction. The obtained dsDNA was directly amplified into ChIP-seq libraries using SeqAmp DNA Polymerase and the forward and reverse primers from the Indexing Primer Set HT for Illumina followed by size selection using AMPure XP beads (Beckman Coulter), and 100-bp paired-end sequenced using a NovaSeq 6000 (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Quality control of sequenced reads was assessed by FastQC (version 0.11.9). Cutadapt (version 4.1) was used to remove adapters sequences.Trimmed sequenced reads were aligned to the mouse reference genome (mm10) using Bowtie2 (version 2.3.4.1) and filtered with samtools (version 1.15.1).
Normalized coverage tracks (CPM normalization in bigWig format) were generated with bamCoverage from deepTools (version 3.4.2). The Integrative Genomic Viewer (version 2.10.2) (https://software.broadinstitute.org/software/igv/) was used to visualize peaks.
csaw (R package, version 1.28.0 was used to detect differentially bound sites (DBS) with sliding windows. Problematic regions in the mouse genome (mm10) were discarded using a curated blacklist downloaded from https://sites.google.com/site/anshulkundaje/projects/blacklists. Reads overlapping a sliding window across the genome were counted with windowCounts. The function normFactors was used to remove compositional biases and to perform count normalization. The resulting matrix of window's counts was analyzed with the voom method from the limma package.DBS were identified using a linear model with background (WT and KO) as predictor. Significant DBSs were selected with FDR <0.05.
Assembly: mm10
Supplementary files format and content: .bw
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| Submission date |
Jul 18, 2023 |
| Last update date |
May 30, 2024 |
| Contact name |
Xin Sun |
| E-mail(s) |
sunxin@ifrec.osaka-u.ac.jp
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| Organization name |
IFReC, Osaka University
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| Street address |
3-1 Yamadaoka, Suita
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| City |
Osaka |
| ZIP/Postal code |
5650871 |
| Country |
Japan |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE237640 |
Identification of the genome-wide binding sites of OCT2 in Reg-1fl/fl- and Reg-1ΔNK-NK cells. [ChIP-Seq] |
| GSE237643 |
Deletion of the mRNA endonuclease Regnase-1 promotes NK cell anti-tumor activity via OCT2- dependent transcription of Ifng |
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| Relations |
| BioSample |
SAMN36535415 |
| SRA |
SRX21075957 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7635067_KO-1_ChIP.bw |
26.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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