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| Status |
Public on Jun 26, 2024 |
| Title |
Sample 45: HT1376-TetOn-ARID1A_DMSO_without Dox_IgG_R1 |
| Sample type |
SRA |
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| Source name |
HT1376
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HT1376
treatment1: DMSO
treatment2: without Dox
cell type: HT1376-TetOn-ARID1A
target: IgG
repeat: R1
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| Treatment protocol |
HT1376-TetON control cells and a clonal HT1376-TetON-ARID1A cell line were induced with 50 ng/ml doxycycline for 24 hours and then treated for 4 days with DMSO or 250 nM CPI-0209.
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| Growth protocol |
Cells were grown in MEM(Earl's Salts) + 10% FBS, 1% Non-essential AA, 1 mM NaPyruvate, 1% Pen/Strep
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed by adding formaldehyde to final concentration of 0.1%. Fixation was done for 1 min at room temperature. Crosslinking was stopped by adding glycine to a final concentration of 125 mM. The fixed cells were snap-frozen and shipped by Epicypher (Durham, NC) for CUT&RUN processing. 0.5x106 fixed cells were washed twice in 100 uL Wash Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Protease Inhibitors) before resuspending in 100 uL Wash Buffer and transfer to tubes containing 10 uL of activated ConA bead (Epicypher #21-1401). The ConA beads absorbed cells were resuspend in 50 uL Antibody Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.02% Digitonin, 2 mM EDTA, 1x Protease Inhibitors) containing 0.5 ug of antibodies recognizing ARID1A (BETHYL A301-041A), SMARCA4 (BETHYL A300-813A), H3K4me3 (Epicypher #13-0041), H3K27me3 (Epicypher #13-0030), H3K27ac (Epicypher #13-0045) or IgG (Epicypher #13-0041). The beads and antibodies were incubated overnight at 4C before washing with 250 uL Digitonin Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, 1x Protease Inhibitors) twice. The beads were incubated with 50 uL cold Digitonin Buffer with 2.5 uL CUTANA pAG-MNase (Epicypher # 15-1016) for 10 min at room temperature before washing with 250 uL cold Digitonin Buffer twice. The washed beads were incubated with 50 uL Digitonin Buffer with 1 uL 100 mM CaCl2 for 2 hr at 4C.
The released DNA were purified by NEB Monarch DNA Cleanup Kit (NEB T1030S) and subjected for library preparation using NEB Ultra II Library Kit (NEB E7645S) following the manufacturer’s instructions. Prior to sequencing, library size and concentration were assessed by Agilent High Sensitivity DNA ChIP (Agilent 5067-4626) on 2100 Bioanalyser (Agilent). Sequencing was performed with an Illumina MiniSeq or NextSeq 500 platforms to generate 3-5 million paired-end reads.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: Cut&Run
Sequence reads were mapped to the genome using the Bowtie2 algorithm with setting as “–local –very-sensitive-local –no-unal –no-mixed –no-discordant –phred 33 -I 10 -X 700”.
Duplicated reads were removed using Picard.
For comparative analysis, E.coli spike-in protocols and normalization were used, the number of test tags are adjusted by a factor that would result in the same number of usable spike-in E.coli tags for each sample within a normalization group.
Peaks were called using the MACS2 algorithms and the signal from IgG was used as control. MACS2 default cutoff is pvalue 1e-5 for both narrow peaks and broad peaks.
Genome_build: hg38
Supplementary_files_format_and_content: bw file: bigwig; narrowPeak/broadPeak: called peaks
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| Submission date |
Jun 09, 2021 |
| Last update date |
Jun 26, 2024 |
| Contact name |
Patrick Trojer |
| E-mail(s) |
patricktroj@gmail.com
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| Organization name |
Constellation Pharmaceuticals
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| Street address |
215 First Street, Suite 200
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| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE176477 |
Epigenomic change of ARDI1A mutant bladder cancer cell line treated with CPI-0209 or induced ARID1A expression |
| GSE176493 |
Comprehensive target engagement by EZH2 inhibitor CPI-0209 allows for preferential targeting of ARID1A mutant urothelial carcinoma |
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| Relations |
| BioSample |
SAMN19643501 |
| SRA |
SRX11101542 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5366213_EpiSoW2007_18_IgG_FTcXL_HT1376_4a_500k_K4met2Ec_S4_sf0.80b20s100dt.bw |
171.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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