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| Status |
Public on Sep 06, 2022 |
| Title |
Rh30_DMSO-IgG-Rep1_CaR090121_hg19 |
| Sample type |
SRA |
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| Source name |
RH30
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| Organism |
Homo sapiens |
| Characteristics |
cell line: RH30
treatment: DMSO
chip antibody: IgG (Epicypher 13-0042)
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| Growth protocol |
RH30 cells were cultured with RPMI 1640 media supplemented with 100 U/mlpenicillin/streptomycin and 10% fetal bovine serum. Cells were maintained at 37 °C in an atmosphere of 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The CUT&RUN library DNAs of Rh30 with and without QC6352 200 nM treatments were prepared by following the protocol as described previously 6 with minor modifications. Briefly, 200,000 Rh30 cells treated with or without QC6352 were washed 3 times with CUT&RUN washing buffer (20 mM HEPES at pH 7.5, 150 mM NaCl, 0.5 mM spermidine (S2501-5G, Sigma-Aldrich), 1× protease inhibitor cocktails (11836170001, Roche) and then transferred to a 1.5-ml tube containing 50 µL washing buffer. Cells were collected with concanavalin A-coated beads (BP531, Bangs laboratories) and incubated with antibodies (1:100), normal rabbit IgG (CS200581, Millipore), rabbit anti-KDM4B (A301-478A, Bethyl Laboratories) and mouse anti-PAX3-FOXO1 (a gift from Dr. Taosheng Chen) overnight at 4 °C in the antibody incubation buffer (20 mM HEPES at pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1× protease inhibitor cocktails, 2 mM EDTA and 0.02% digitonin). The samples with mouse anti-PAX3-FOXO1 antibody were washed 3 times with washing buffer and incubated with rabbit anti-mouse secondary antibody (ab6709, Abcam) overnight at 4 °C. After unbound antibodies were washed away, 140 μg/ml protein A-MNase (pA-MN; a gift from Dr. Steven Henikoff) was added at a 1:280 ratio and incubated for 1 hr at 4 °C. After washing, 3 μl of 100 mM CaCl2 was added to activate pA-MN for 30 min at 4 °C and the reaction was stopped by adding the same volume of 2× STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% digitonin, 100 µg/ml RNase A and 50 µg/ml glycogen). The protein–DNA complexes were released by 10-min incubation at 37°C followed by centrifugation at 16,000 g for 5 min at 4 °C. After transferring the supernatant to a new 1.5 ml Eppendorf tube, 2 μl of 10% (wt/vol) SDS and 1.5 μl of proteinase K (20 mg/ml) were added and incubated for 1 hr at 50 °C. DNA was then extracted with phenol/chloroform/isoamylalcohol followed by ethanol precipitation with glycogen and then dissolved in water. Sequencing libraries were prepared using an Accel-NGS 1S plus DNA library preparation kit (10096, Swift Biosciences) and Accel-NGS 1S plus combinatorial Dual Indexing Kit (18096, Swift Biosciences) for Illumina platforms following the manufacturer's instructions. The PCR products were cleaned with SPRIselect beads and quantified using Qubit dsDNA HS assay kit (Q32854, Thermo fisher). The libraries were sequenced on a HiSeq2500 with paired-end 50-bp reads (Illumina).
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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
Fastq files were demultiplexed using illumine local run manager, then trimmed 3’ raw reads with cutadapt (v1.2.1) using Trim_Galore (v0.6.6). Quality trimmed reads were mapped to both hg19 and E. coli K12, MG1655 for normalization purpose by bwa (v0.7.17) and converted to bam file by samtools (v1.9). Uniquely mapped paired reads were extracted by samtools and sorted by biobambam2 (v2.0.87), then bedtools (v 2.17.0) were used to convert bam to bedpe file. Fragments size shorter than 2kb were extracted and center 80bp of each fragment were used to generate bigwig track file by UCSC tools(v4). For pairwise comparisons, E. coli normalization is included to generate bigwig file. Calculated the ratio using E. coli spike-in reads to unique aligned reads for each sample, then count scale factor due to after normalization the E. coli spike-in signal is equal across all samples. MACS2 (v2.1.1) is used to call narrow peaks across all samples.
Assembly: hg19
Supplementary files format and content: bigwig,narrowPeak
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| Submission date |
Mar 16, 2022 |
| Last update date |
Sep 06, 2022 |
| Contact name |
Hongjian Jin |
| E-mail(s) |
hongjian.jin@STJUDE.ORG
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| Organization name |
St Jude Children's Research Hospital
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| Department |
Center for Applied Bioinformatics
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| Street address |
262 Danny Thomas Place
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| City |
Memphis |
| State/province |
TN |
| ZIP/Postal code |
38015 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE157095 |
Targeting KDM4B to disrupt the core regulatory transcription network governed by PAX3-FOXO1 in high-risk rhabdomyosarcoma |
| GSE198753 |
Targeting KDM4 for treating PAX3-FOXO1-driven alveolar rhabdomyosarcoma [CUT&RUN, 2] |
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| Relations |
| BioSample |
SAMN26722576 |
| SRA |
SRX14473839 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5956321_Rh30_DMSO-IgG-Rep1_CaR090121_hg19.sort.bw |
56.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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