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| Status |
Public on Mar 28, 2024 |
| Title |
Treg Ikzf1 conditional knockout ex vivo 2 Foxp3_chip |
| Sample type |
SRA |
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| Source name |
Children’s Hospital of Philadelphia
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| Organism |
Mus musculus |
| Characteristics |
tissue: Tregs derived from spleen
antibody: Foxp3
genotype: Ikzf1 conditional knockout
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5x10^6 purified cells were fixed in medium for 15 minutes at room temperature using complete cell fixative solution prepared using formaldehyde and cell fixative solution from the kit. The reaction was stopped by adding 1/20 media volume of stop solution, cells were washed with ice-cold PBS and a cell pellet was stored at -80C for later use or cells were lysed in chromatin preparation buffer cells as described in the protocol. The cell pellet was resuspended in ChIP buffer and chromatin was sonicated by a QSonica Q800R sonicator with settings: amplitude 20%, pulse for 30 seconds on, 30 second off, for a total of 30 cycles. For input DNA preparation, 25 ul of the sonicated sample was removed and DNA was isolated as suggested in the protocol. An agarose (1%) gel electrophoresis was performed for DNA isolated from the input fraction to determine the sonication efficiency. 5x106 purified cells were fixed in medium for 15 minutes at room temperature using complete cell fixative solution prepared using formaldehyde and cell fixative solution from the kit. The reaction was stopped by adding 1/20 media volume of stop solution, cells were washed with ice-cold PBS and a cell pellet was stored at -80C for later use or cells were lysed in chromatin preparation buffer cells as described in the protocol. The cell pellet was resuspended in ChIP buffer and chromatin was sonicated by a QSonica Q800R sonicator with settings: amplitude 20%, pulse for 30 seconds on, 30 second off, for a total of 30 cycles. For input DNA preparation, 25 ul of the sonicated sample was removed and DNA was isolated as suggested in the protocol. An agarose (1%) gel electrophoresis was performed for DNA isolated from the input fraction to determine the sonication efficiency. 5x106 purified cells were fixed in medium for 15 minutes at room temperature using complete cell fixative solution prepared using formaldehyde and cell fixative solution from the kit. The reaction was stopped by adding 1/20 media volume of stop solution, cells were washed with ice-cold PBS and a cell pellet was stored at -80C for later use or cells were lysed in chromatin preparation buffer cells as described in the protocol. The cell pellet was resuspended in ChIP buffer and chromatin was sonicated by a QSonica Q800R sonicator with settings: amplitude 20%, pulse for 30 seconds on, 30 second off, for a total of 30 cycles. For input DNA preparation, 25 ul of the sonicated sample was removed and DNA was isolated as suggested in the protocol. An agarose (1%) gel electrophoresis was performed for DNA isolated from the input fraction to determine the sonication efficiency. For all ChIP-seq experiments, 3 biological replicates of cells isolated from 3 individual mice were used. Chromatin immunoprecipitations were performed using ChIP-IT high-sensitivity kits (cat #53040, Active Motif) following the manufacturer’s instructions. Briefly, 5x106 purified cells were fixed in medium for 15 minutes at room temperature using complete cell fixative solution prepared using formaldehyde and cell fixative solution from the kit. The reaction was stopped by adding 1/20 media volume of stop solution, cells were washed with ice-cold PBS and a cell pellet was stored at -80C for later use or cells were lysed in chromatin preparation buffer cells as described in the protocol. The cell pellet was resuspended in ChIP buffer and chromatin was sonicated by a QSonica Q800R sonicator with settings: amplitude 20%, pulse for 30 seconds on, 30 second off, for a total of 30 cycles. For input DNA preparation, 25 ul of the sonicated sample was removed and DNA was isolated as suggested in the protocol. An agarose (1%) gel electrophoresis was performed for DNA isolated from the input fraction to determine the sonication efficiency. ChIP-validated anti-mouse Ikaros antibody (cat #39355) and H3K27Ac antibody (cat #39133) were purchased from Active Motif. For Foxp3 ChIP-seq, eBioscience anti-mouse monoclonal antibody (cat#14-5773-82) was purchased from ThermoFisher Scientific.
The volume of the sheared chromatin was adjusted to 200 ul using ChIP buffer, and to which was added 5 ul of Protease inhibitor, and a mix containing ChIP antibody (4 ug) and 5 ul of blocker, mixed and pre-incubated at room temperature for a minute. Final volume of the ChIP reaction was 240 ul, which was incubated at 4C overnight on a rotator. Antibody precipitated chromatin immune complexes were collected using washed protein G agarose beads and immune complexes were washed 5X in ChIP filtration columns using wash buffer, and eluted the DNA with elution buffer. The eluted DNA was reverse cross linked and further purified through DNA purification columns. ChIP’d DNA was eluted from the column using 30 ul of DNA purification elution buffer. All ChIP-seq and input DNA libraries were made using ThruPLEX DNA-Seq kit (cat #R400674, Takara Bio, USA) following the manufacturer’s instructions. In brief, the fragmented DNA obtained from ChIP reaction or input DNA was end repaired to generate blunt ends, to which stem-loop adaptors with blocked 5’ ends are ligated. Libraries were amplified through high fidelity amplification buffer mix and Takara dual indexing primers (cat# R400407). Finally, the amplified dual indexed libraries were purified using AMpure XP beads (Beckman Coulter, Cat # A63880) at 1:1 ratio. The purified DNA was recovered from the beads using 20 ul of TE buffer. The library quality was checked on a bioanalyzer using high sensitivity DNA Chip. Library DNA concentration was determined using Qubit. Dual-indexed ChIP-seq libraries were pooled and sequenced on a the Illumina NovaSeq 6000 platform.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Foxp3_IKcKO_CHIP_2
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| Data processing |
Bases were called using Illumina bcl2fastq2 conversion v2.29
Reads were aligned to mm9 using bowtie2 and duplicated reads were marked using Picard with parameters VALIDATION_STRINGENCY=LENIENT and ASSUME_SORTED=true and removed samtools.
Peaks were called using MACS2 with the parameters -g mm9 –nomodel –p 0.01 –keep-dup_all with the – extsize estimated fragment size from the strand cross correlation for each replicate of H3K27ac, Ikzf1, or Foxp3 with matching input sample.
Within condition peaks were filtered to ones found in at least two replicates.
bigwig files were generated using deeptools (v 3.30) bamCoverage function with the parameters --normalizeUsing RPGC --effectiveGenomeSize 2304947926 --binSize 10 --extendReads
Assembly: mm9
Supplementary files format and content: bigwig files
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| Submission date |
Apr 05, 2022 |
| Last update date |
Mar 28, 2024 |
| Contact name |
Matthew C Pahl |
| E-mail(s) |
pahlm@email.chop.edu
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| Phone |
5704282303
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| Organization name |
Children's Hospital of Philadelphia
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| Department |
Center for Spatial and Functional Genomics
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| Lab |
Struan Grant and Andrew Wells
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| Street address |
3615 Civic Center Blvd
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE200177 |
Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function [ChIP-Seq] |
| GSE200179 |
Foxp3 depends on Ikaros for control of regulatory T cell gene expression and function |
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| Relations |
| BioSample |
SAMN27306006 |
| SRA |
SRX14743589 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6019849_Foxp3_IKcKO_CHIP_2.bigwig |
41.7 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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