 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 19, 2024 |
| Title |
PROTAC experiment, KELLY cells, GSK6991, H3K9ac antibody, 6 hours, replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
KELLY
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: KELLY
cell type: Neuroblastoma, MYCN amplified
chip antibody: H3K9ac (Abcam cat.# ab4441)
spike-in antibody: spike-in antibody (Active Motif cat.#61686)
|
| Treatment protocol |
Antibodies used for ChIP were HA-tag (Cell Signaling Technologies, #3724), H3K9ac (Abcam #ab4441), H3K27ac (Abcam #ab4729), and MYCN (Abcam #ab16898). For HA-tag ChIP in KELLY, 5 µg of HA antibody was used for 20 million cells. For NGP and NB1 HA-tag ChIPs, 50 µl of HA antibody was used for 20 million cells. For MYCN, 10 µg of antibody was used per sample of 20 million cells. For H3K9ac, 5 µg of antibody was used per sample of 5 million cells. For H3K9ac, 7.5 µl of spike-in chromatin was used per sample. For MYCN, 3 µl of spike-in chromatin was used per sample. For HA ChIP, 10 µl of spike-in chromatin was used per sample of 20 million cells. For H3K27ac, 5 µg antibody and 2 µl of spike-in chromatin was used for 10 million cells. For each sample, protein A or protein G beads (Life Technologies #10003D) were prepared by washing three times in BSA blocking solution, then resuspended in 250 µl/sample BSA blocking solution with appropriate antibody and spike-in antibody. Beads were incubated overnight with rotation, washed four times with BSA blocking solution, and resuspended in 100 µl/sample BSA blocking solution. Spike-in chromatin was added to the beads two hours before adding sheared human chromatin and incubated with rotation at 4C.
|
| Growth protocol |
Neuroblastoma cell lines were grown in 15 cm plates.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
20 million cells were cross-linked with 1% formaldehyde in 22 mL warm media for 10 minutes with rotation followed by quenching with the addition of 1.1 mL of 2.5 M glycine for 5 minutes. Cells were pelleted by centrifugation at 600xg and resuspended in 1 mL cold PBS supplemented with protease inhibitors (Thermo Fisher # 78430) and 1 mM PMSF (Sigma Aldrich #P7626). This wash was repeated twice for a total of three washes in PBS. Cells were resuspended in cytoplasmic and then nuclear lysis buffer, and cells were sheared on an E220 Covaris sonicator (Duty cycle: 5%; Peak power: 140W; Cycles per Burst:200; Temp: 4C; time 15 min). Sheared lysates were cleared by centrifugation at 15,000g for 10 minutes at 4C and incubated overnight with rotation at 4C with magnetic beads prebound with antibody, spike-in antibody (Active Motif #61686), and spike-in chromatin (Active Motif #53083). Following overnight incubation, beads were washed twice with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tri-HCl pH8.1, 150 mM NaCl), twice with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tri-HCl pH8.1, 500 mM NaCl), twice with LiCl wash buffer (0.25 M LiCl, 1% IGEPAL-CA 630, 1% deoxycholic acid, 10 mM Tri-HCl ph8.1, 1 mM EDTA), and once with TE. DNA was eluted in elution buffer (1% SDS, 0.1 M NaHCO3). Cross linking was reversed overnight at 65C with 0.9% RNAse A, 0.9% Proteinase K, and 0.182 M NaCl. DNA was isolated by adding Agencourt AMPure XP beads (Beckman Coulter A63880) at 1.2x volume, incubating 20 minutes, washing twice with 80% ethanol, and eluting into 10-20 µl TE.
ChIP-Seq libraries were prepared using Swift S2 Acel reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 1ng of DNA according to manufacturer’s protocol and 14 cycles of PCR amplification. Finished sequencing libraries were quantified by Qubit fluorometer and Agilent TapeStation 2200. Subsequently, libraries were paired end sequenced on Illumina NovaSeq 6000.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Sequencing performed at Novogene Co
|
| Data processing |
ChIP-Seq data analysis was performed in alignment with the ENCODE Consortium standards (https://www.encodeproject.org/chip-seq/). Quality control tests for unmapped sequences were performed by using the FastQC v.0.11.9 software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All samples passed the QC, except MYCN replicate 2 for the PROTAC experiment on KELLY cells treated with DMSO at 6h. The MYCN replicate 2 for the PROTAC experiment on KELLY cells treated with DMSO at 6h was removed.
The ChIP-sequences were aligned to the GRCh38/hg38 genome and to the spiked-in dm6 Drosophila melanogaster genome using bowtie2-2.3.5 (Langmead and Salzberg, 2012) with the standard options. PCR duplicates were removed with the Picard Mark Duplicates method implemented in the sambamba v0.7.1 tool (Tarasov et al., 2015). The alignments with MAPQ < 5 were skipped.
The mapped reads were normalized in units of reads per kilobase per million (RPKM or rpm/bp) and coverage tracks for the RPKM signal were created as bigwig files for bins of size 20 base pairs by using the bamCoverage tool available in deepTools v3.5.1 (Ramírez et al., 2016). The Active Motif Spike-in Normalization protocol was then applied to each hg38 sample. The dm6 genome-wide counts distribution on 200 bp size bins was computed with the multiBamSummary function available from the deepTools v3.5.1. The dm6 uniquely mapped reads were summed up from all the bins with at least 10 mapped dm6 reads. Each hg38 sample was normalized by multiplying the human tag counts with the scaling factor derived from the Drosophila reads as ratio between the average of the uniquely mapped dm6 counts across all samples vs. the dm6 uniquely mapped reads counts for that sample.
Peak calling was performed against the input control using the model-based MACS2 v2.1.1.20160309 software (Zhang et al., 2008), with the significance cut-off FDR ≤ 0.01. Area under the curve (AUC) RPKM normalized signal across genomic regions was computed with the bwtool software (Pohl and Beato, 2014). Each set of MACS2 peaks was curated by removing the binding regions with low area under curve RPKM coverage (log2(AUC+1)< 13) and the regions overlapping with the ENCODE black-listed regions for hg38 (available at https://www.encodeproject.org/annotations/ENCSR636HFF/). Quality control tests for the peaks were performed by using the ChIPQC library available from Bioconductor v3.9 (Carroll et al., 2014).
The peaks were annotated with the closest hg38 genes by using the annotatePeaks function implemented in the Homer v4.11 platform (Heinz et al., 2010) and the GREAT annotation platform (McLean et al., 2010). Binding peaks and normalized binding signal were visualized on the Integrative Genomic Viewer (IGV) v2.12.3 platform (Thorvaldsdóttir et al., 2013). Gene promoter regions were defined as the +/- 3.0 kb intervals around the hg38 gene transcription start site (TSS). Enhancer regions were defined as the H3K27ac binding regions outside TSS +/- 3.0 kb gene promoter regions. The samtools v1.9 (Li et al., 2009) and the BEDTools v2.29 suite (Quinlan and Hall, 2010) were used to perform various mapping and genomic region analyses (indexing, sorting, intersection, merging).
Per each antibody, the binding sites identified by MACS2 for treatment and control conditions across time points were merged into the antibody’s set of aggregated peaks. A peak by sample counts matrix was created by counting the reads overlapping each aggregated peak with the multiBamSummary tool available from deepTools. The counts matrix was used to perform differential peak analysis. For a specific genomic region, the changes in signal between two conditions were classified as increase, decrease or not significant based on the absolute cut-off 1.5 for the delta area under curve scores; the significance was quantified based on the cut-off p-value ≤ 0.10 for the differences in the mapped reads by using edgeR v3.36.0 with the glmLRT model (Robinson et al., 2010). Heatmaps of AUC ChIP-seq normalized signal occupancy on genomic regions were created using the computeMatrix and the plotHeatmap tools available in deepTools v3.5.1. The plotProfile tool from deepTools v3.5.1 was used to create metaplots based on the average normalized scores across genomic regions. Differences between metaplots were estimated based on the t-test with Welch correction (cutoff P-value ≤ 0.05) for the area under curve (AUC) RPKM normalized signal across the regions for the conditions. Motif enrichment analysis was performed for genomic regions and gene promoters using the Homer v4.11 platform (Heinz et al., 2010) and the MEME Suite v5.4.1 platform (FIMO method) (Bailey et al., 2015) with the adjusted p-value cutoff ≤ 0.01.
Assembly: hg38
Supplementary files format and content: bigWig RPKM binding signal
|
| |
|
| Submission date |
Oct 30, 2023 |
| Last update date |
Apr 19, 2024 |
| Contact name |
Gabriela Alexe |
| E-mail(s) |
[email protected]
|
| Organization name |
Broad Institute
|
| Department |
Computational Biology and Bioinformatics
|
| Street address |
415 Main St.
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE211355 |
The SAGA complex maintains the oncogenic gene expression program in MYCN-amplified neuroblastoma |
| GSE211953 |
The SAGA complex maintains the oncogenic gene expression program in MYCN-amplified neuroblastoma [ChIP-Seq] |
|
| Relations |
| BioSample |
SAMN38043885 |
| SRA |
SRX22290441 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
