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| Status |
Public on Jul 07, 2020 |
| Title |
nTreg_ChIPseq_dBRD9_Foxp3 |
| Sample type |
SRA |
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| Source name |
nTregs isolated from Foxp3Thy1.1/Rosa-Cas9 mice
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| Organism |
Mus musculus |
| Characteristics |
genotype/variation: Foxp3Thy1.1/Rosa-Cas9
cell type: nTregs
treatment: 2.5 uM dBRD9
guide rna: none
chip antibody: In-house made Foxp3 antibody
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ATAC-seq, 50k nTregs per replicate were washed with cold PBS and resuspension buffer (RB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) before lysis in RB with 0.1% NP40. Cells were then incubated in transposition mix containing Tn5 transposase for thirty minutes at 37degC. Purified DNA was ligated with adapters (NuGEN), amplified and size selected for sequencing.
For ChIP-Seq, 7.5e6 nTregs were collected and cross-linked first in 3mM disuccinimidyl glutarate (DSG) then in 1% formaldehyde. After quenching the excess formaldehyde with 125 mM glycine, the fixed cells were washed, pelleted and flash-frozen. Upon thawing, the cells were resuspended in lysis solution (50 mM HEPES-KOH pH 8, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100 and incubated on ice for ten minutes. The isolated nuclei were washed with wash solution (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl) and shearing buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl pH 8) then sheared in a Covaris E229 sonicator for 10-20 minutes to generate DNA fragments between ~ 200-1000 bp. After clarification of insoluble material by centrifugation, the chromatin was immunoprecipitated overnight at 4C with antibodies against Foxp3, BRG1, BRD9, PHF10 and H3K27ac then bound to Protein A+G Dynabeads (Invitrogen) in ChIP buffer (50 mM HEPES-KOH pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% DOC, 0.1% SDS). Antibody bound DNA were washed and treated with Proteinase K and RNase A and the purified ChIP DNA was used for library generation for subsequent sequencing. ChIP-Seq libraries were generated using the NuGen Ovation Ultralow Library System V2 following the manufacturer's instructions.
For RNA-seq, total RNA was isolated using Quick-RNA Miniprep Kit (Zymo Research). RNA-Seq libraries were prepared using Illumina TruSeq RNA Library Prep Kit v2 following the manufacturer's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
ChIP-seq for Foxp3 in nTregs treated with dBRD9.
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| Data processing |
Basecalls performed using RTA (Illumina).
Reads were aligned to the mouse genome (mm10) using STAR alignment tool (v2.5) for RNA-seq, ChIP-seq and ATAC-seq. In all cases, only reads that mapped to a unique genomic locations (MAPQ > 10) were used for downstream analysis.
HOMER (v4.8) (http://homer.salk.edu/homer/) was used to process alignment files to generate ChIP-seq bed files. ChIP-seq peaks for Foxp3, BRD9, BRG1 and PHF10 were found by using the findPeaks program in HOMER with the parameter "-style factor" versus the appropriate ChIP input experiments as background. ChIP-seq peaks for H3K27ac were called using the parameter "-style histone." ATAC-seq peaks were called using the parameter "-style dnase." For RNA-Seq, Reads Per Kilobase of exon per million mapped reads were calculated using HOMER (http://homer.salk.edu/homer/) across annotated gene exons (RefSeq).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Bed file Columns: (1) peak ID (2) chrom (3) chromStart (4) chromEnd (5) strand.
Supplementary_files_format_and_content: DiffExpOutput tab-delimited text file Columns: (1) RefSeq acc (2) chr (3) start (4) end (5) strand (6) length (7) copies (8) annotation (9-12) FPKM for each experiment (13) Log2 fold change (14) p value (15) adjusted p value.
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| Submission date |
Apr 15, 2019 |
| Last update date |
Nov 17, 2022 |
| Contact name |
Diana C. Hargreaves |
| E-mail(s) |
dhargreaves@salk.edu
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| Organization name |
The Salk Institute for Biological Studies
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| Street address |
10010 North Torrey Pines Road
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| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE129846 |
A genome-wide CRISPR screen reveals a role for the non-canonical nucleosome remodeling BAF complex in Foxp3 expression and regulatory T cell function |
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| Relations |
| BioSample |
SAMN11421771 |
| SRA |
SRX5689487 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3723370_nTreg_ChIPseq_dBRD9_FOXP3_F7peaks.bed.gz |
98.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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