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| Status |
Public on Jul 08, 2024 |
| Title |
H3K4me3, Dox, rep1 |
| Sample type |
SRA |
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| Source name |
4D6
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| Organism |
Homo sapiens |
| Characteristics |
cell line: 4D6
cell type: thymic epithelial cells
genotype: WT AIRE
treatment: Dox 24hr
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| Treatment protocol |
4D6 stable cells were seeded on 150mm plates with or without Dox and grown for 24 hrs. For drug-treated samples, equal volume of DMSO, 3 µm A-485 (Tocris Bioscience), or 0.25 µm dCBP-1 (MedChemExpress) was added to cells 4 hrs prior to harvest.
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| Growth protocol |
4D6 cells were maintained in RPMI supplemented with 10% FBS, 1% L-glutamine
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For anti-FLAG (AIRE) and anti-p300 ChIP-seq, cells were washed 3 times with PBS and then crosslinked with 2mM Disuccinimidyl glutarate (DSG) in PBS for 45 minutes at room temperature. Cells were then washed again 3 times with PBS and then crosslinked with 1% formaldehyde (Sigma, Thermofisher, and Electron Microscopy Sciences) in PBS for 10 minutes at room temperature. For anti-histone mark ChIP-seq, cells were crosslinked with 1% formaldehyde in fresh media for 10 minutes at room temperature. After formaldehyde crosslinking, all cells were washed 1 time with PBS and quenched with 0.125 M glycine in PBS for 5 min at room temperature. Quenched cells were washed with ice cold PBS, then harvested in ice cold PBS supplemented with 0.5 mM PMSF. Cells were spun down at 500 g for 5 minutes and cell pellets were supplemented with 1 µl of 100mM PMSF and 1 µl 1X mammalian protease inhibitor cocktail (G-Biosciences), then flash frozen in liquid nitrogen and stored at -80°C until ready to use. For one ChIP pull-down, ~15x106 cells were used. Cells pellets were thawed on ice for 15 minutes and then resuspended in ice-cold One-Step Lysis Buffer (50 mM Tris H 7.5, 1% SDS, 0.25% Sodium Deoxycholate and 1 X mammalian protease inhibitor cocktail). Cells were incubated on ice for 10 min and then 700 µl ChIP Dilution Buffer (50 mM Tris pH 7.5, 0.01% SDS, 150 mM NaCl, 0.25% Triton-X, 1 X mammalian protease inhibitor cocktail) was added. Chromatin was sheared using with a Covaris M220 ultrasonicator (settings: 5% duty factor, 75W max power, 200 cycles per burst, 20 min) at 6°C. Lysates were spun down in a refrigerated centrifuge for 10 minutes at 18,000 g. Cleared lysate was 3-fold diluted with ChIP Dilution Buffer, spike-in chromatin (600ng per pull-down, Active Motif) was added when cells were treated with p300 inhibitors or DMSO, and 2-3% of the input was saved for later use. Lysates were nutated with antibodies [for each pull-down when indicated, 2.5 µg of anti-FLAG (M2, Sigma); 5 µg anti-H3K4me1, 5 µg anti-H3K27me3, 4 µg of anti-p300 (D2X6N, Cell Signaling Technologies); 4 µg of anti-H3K27Ac (Cell Signaling Technologies); 4 µg of anti-H3K4me0 (Active Motif); an additional 2 µg spike-in antibody (Active Motif) was included when cells had been treated with p300 inhibitors or DMSO] for 16 hrs at 4°C. Protein-DNA complexes were immunoprecipitated using Protein G magnetic beads (Active Motif) with 2 hrs nutation at 4°C. Protein G beads were washed with the following ice-cold buffers: RIPA buffer (0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X-100, 1 mM EDTA, 10 mM Tris pH 8, 150 mM NaCl); RIPA supplemented with 350 mM NaCl; LiCl Buffer (10 mM Tris pH 8, 250 mM LiCl, 0.5% Triton X-100, 0.5% Sodium Deoxycholate) and Tris Buffer (10 mM Tris pH 8.5). Protein-DNA complexes were eluted with Elution Buffer (10 mM Tris pH 8, 1 mM EDTA, 0.1% SDS, 150 mM NaCl, 5 mM DTT) with gentle agitation at 65°C for 1 hr. Reserved ChIP inputs were diluted two-fold with Elution Buffer. Dilute inputs and eluted protein-DNA were treated with RNAse (Machery-Nagel) at 37°C for 30 min, then Proteinase K (New England Biolabs) at 65°C for 16 hrs to ensure reverse crosslinking of DNA. Reverse cross-linked DNA was purified using SPRI Select beads (Beckman).
ChIP-seq DNA libraries were prepared using NEBNext Ultra II DNA library Prep Kit for Illumina (New England Biolabs) according to manufacturer’s protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
H3K4me3_Dox_PD1
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| Data processing |
QC was performed on demultiplexed sequencing files using FASTQC (v0.11.3). Sequencing reads were trimmed using Trimmomatic (v0.36) and aligned to reference genome (GRCh38 primary assembly) using bwa (v0.7.17). The resulting SAM files were converted to BAM files, sorted and indexed using Samtools (v1.6). Samples with drosophila spike-in chromatins were aligned to a customized reference genome consisting both hg38 and dm6, and the sorted and index BAM files were subsequently split into separate BAM files containing hg38- and dm6-mapped reads, respectively, using plit_bam.py module in the SPIKER tool. hg38- and dm6-mapped reads were deduplicated using the alignmentSieve function in deepTools (v3.5.1), and unique dm6-mapped reads were counted to calculate the scaling factors.
Peak calling was performed using MACS2 (v2.2.7.1) with the following parameters: macs2 callpeak -f BAMPE -B -g 3.2e+9 --keep-dup 1 --SPMR --nomodel --extsize 250 -q 0.05 --cutoff-analysis. When calling peaks in WT and mutant AIRE ChIP-seq samples, corresponding input controls were included to remove the effect of background noise.
WT and mutant AIRE ChIP-seq BAM files and peaks were imported into DiffBind (v3.8.4), and a census of all AIRE peaks (n=13023, summits +/– 200 bp) were obtained after removing blacklisted and greylisted regions using dba.blacklist and dba.peakset functions. Trimmed Mean of M-values (TMM)-normalized counts-per-million (CPM) reads9 within each peak (summits +/– 200 bp) were obtained using the dba.count function in DiffBind with options.
Peaks called from H3K27ac ChIP-seq (–AIRE expression) were removed of blacklisted regions and subjected to Ranking Ordering of Super Enhancer (ROSE) analysis to obtain putative super-enhancer regions, using 12.5 Kb stitching distance and 2 Kb TSS exclusion parameters.
BAM files were converted to bigwig files using deepTools (v3.5.1). After verification of consistency between replicates, bigwig files were averaged using WiggleTools (v1.2.2) and bedGraphToBigWig (v366) and imported into IGV (v2.15.1) for visualization at specific loci or into deepTools (v3.5.1) for generation of heatmaps and average profiles using the computeMatrix, plotHeatmap and plotProfile functions.
Assembly: hg38, dm6
Supplementary files format and content: bigwig files contain ChIP signals for each sample
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| Submission date |
May 15, 2024 |
| Last update date |
Jul 08, 2024 |
| Contact name |
Qianxia Zhang |
| E-mail(s) |
qianxia.zhang@childrens.harvard.edu
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| Phone |
9018345955
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| Organization name |
Boston Children's Hospital
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| Street address |
3 Blackfan Circle, RM 3117.16
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE243824 |
Mechanism for controlled assembly of transcriptional condensates by Aire [ChIP-seq] |
| GSE243825 |
Mechanism for controlled assembly of transcriptional condensates by Aire |
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| Relations |
| BioSample |
SAMN41402371 |
| SRA |
SRX24558848 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8268206_WT_Dox_H3K4me3_1.bw |
46.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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