|
| Status |
Public on Feb 27, 2024 |
| Title |
Kidney, Foxp1KspKO mice, Input |
| Sample type |
SRA |
| |
|
| Source name |
Kidney
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Kidney
cell type: kidney tubular cells
genotype: Foxp1_KO
chip antibody: none
|
| Treatment protocol |
The digestive system neutralization with DMEM/F12 contain 10% FBS. After centrifugation the kidney digest was re-suspension then washed through a 40um sieves (FALCON; 352340) to collect kidney tubular cells. The ChIP assay was carried out as previously described.
|
| Growth protocol |
Neonatal littermate control and Foxp1KspKO mice were hypothermic anesthetized then perfused with PBS. The kidneys were quickly removed to a 0.5ml DMEM/F12 solution containing 2 g/L collagenase IV (Sigma; C5138) and Benzonase nuclease (Millipore; E1014, 1:2000), digested for 20 min at 37°C with gentle agitation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
cells were lysed and the chromatin was sheared to a size range of 150 to 300 bp using the sonicator (Diagenode, Bioruptor Pico). For each IP to be performed, aliquot 30μL magnetic Protein G beads (Invitrogen; 1003D) were used to preclear the chromatin, and incubated at 4℃ overnight with the previously characterized Foxp1 polyclonal antibody or control rabbit IgG (2729S; Cell Signaling).
Immune complexes were handled according to the manufacturer’s protocol. Reverse cross-linked immunoprecipitated chromatin was extracted using QIAquick PCR Purification Kit. The library was quantified using the QubitTM dsDNA HS Assay Kit (Invitrogen; Q32851) by QubitTM Fluorometer, inspected with Agilent bioanalyzer and StepOnePlus Real-Time PCR.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
Finput-5
|
| Data processing |
Illumina HiSeq xten/NovaSeq 6000 sequencer
The raw paired-end reads were quality checked by FastQC (https://github.com/s-andrews/FastQC) and trimmed to remove adaptor sequences and low-quality reads using Trimmomatic with the default setting.
ChIP-seq reads were aligned to the reference genome GRCm38.86 using the bowtie2 software and only uniquely and non-duplicate mapped reads were used to perform the downstream analysis.
To examine the reproducibility of the ChIP–seq experiments, Deeptools was used to generate a bigwig format to facilitate visualization of the reads coverage and comparison among different samples including input samples.
The number of reads overlapping a region between 3kb upstream and 3kb downstream of the TSS of each gene were calculated by the R package Rsamtools (https://bioconductor.org/packages/Rsamtools). Promoter counts of each gene were used to identify genes bound by Foxp1 (Pvalue <0.05) using the normalizeChIPtoInput function in the R package edgeR.
Average coverage plots and heatmaps for the binding genes were made using ngs.plot. Differential binding analysis between control and Foxp1-deficent conditions was conducted using the R package edgeR.The motifs analysis was used the regulatory sequence analysis tools (RSAT) peak-motifs. All the bigwig files of ChIP-seq and RiboTag RNA-seq were visualized using the Integrative Genomics Viewer (IGV).
Assembly: mm10
Supplementary files format and content: tab-delimited excel file includes the significant enrichment relative to the control
Supplementary files format and content: bigwig files to view the data
|
| |
|
| Submission date |
Jul 26, 2023 |
| Last update date |
Feb 27, 2024 |
| Contact name |
RENHUA SONG |
| E-mail(s) |
Renhua.song1989@gmail.com
|
| Phone |
61452667663
|
| Organization name |
Centenary Institute
|
| Lab |
Epigenetics and RNA Biology Program
|
| Street address |
Charles Perkins Centre - D17, Level 4 West. The University of Sydney Johns Hopkins Drive
|
| City |
Camperdown |
| State/province |
NSW |
| ZIP/Postal code |
2050 |
| Country |
Australia |
| |
|
| Platform ID |
GPL21273 |
| Series (2) |
| GSE238267 |
Identification of Foxp1 regulating target genes in renal cells using ChIP-seq sequencing |
| GSE238268 |
Foxp1 is required for renal intercalated cell differentiation and acid-base regulation |
|
| Relations |
| BioSample |
SAMN36708658 |
| SRA |
SRX21159325 |
