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| Status |
Public on Sep 22, 2022 |
| Title |
2898_hoxb13_k13ac |
| Sample type |
SRA |
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| Source name |
Prostate Cancer
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| Organism |
Homo sapiens |
| Characteristics |
cell line: C4-2B
cell type: Prostate cancer cell line
treatment: Charcoal stripped FBS
chip antibody: HOXB13-K13ac
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| Treatment protocol |
None
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| Growth protocol |
C4-2B cells were grown in DMEM media with charcoal stripped fetal bovine serum.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation was performed using the ChIP-IT Express (Active Motif) in accordance to the manufacturer’s protocol. For cell lines, cells were grown in T-150 flasks and crosslinked with formaldehyde at 70–80% confluency. Frozen cells were lysed using a Dounce homogenizer, and their chromatin was sheared by sonication to 1-kb fragments. Per IP reaction, up to 25 μg of sheared chromatin was incubated with 4 μg of antibodies and 25 μl of protein G magnetic beads at 4°C overnight. The captured antibody-bound protein/DNA complexes were extensively washed, and chromatin was eluted, reverse-crosslinked, and digested with proteinase K. The collected DNA was purified using a PCR purification kit (Qiagen) followed by qPCR analysis or high-throughput sequencing.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 3000 following the manufacturer's protocols.
During library preparation a unique sequence tag called an index was added onto individual samples. This allows multiplexing all the samples together into a sequencing lane. Samples are run either as a single lane or multiple lanes of either type depending on the number of reads per sample required for your analysis. To maximize for HOXB13 Transcription Factor binding sites in the ChIP sequencing we performed HiSeq3000 which obtains ~350M reads/lane at 1x50 read length. The DNA sequencing data was processed through a new pipeline that was generated and used for the ENCODE project.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
Chromatin
C4-2B_hoxb13_k13ac Rep 1
hoxb13_k13ac
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| Data processing |
Basecalls performed using CASAVA version 1.4.
Reads were demultiplexed and mapped to hg19 using BWA. Potential PCR duplicates have been removed. The corresponding IgG and/or input datasets have been used as background controls. Peaks were identified by MACS2 using the ENCODE-DCC/chip-seq-pipeline2.
Sequencing reads were demultiplexed and processed using the ENCODE Transcription Factor and Histone ChIP-Seq processing pipeline with the “histone” option (https://github.com/ENCODE-DCC/chip-seq-pipeline2, version v1.1.6-10-gd4f6442) (Roadmap Epigenomics et al., 2015), which aligns reads to human genome (hg19) by bowtie2, generates signal strength relative to the control samples and creates the optimal set of peaks using MACS2.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: bigwig files were generated by converting MACS2 output from bedGraph format; Scores represent normalized signal strength.
Supplementary_files_format_and_content: MACS2 peaks in narrowPeak format.
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| Submission date |
Mar 17, 2021 |
| Last update date |
Sep 22, 2022 |
| Contact name |
Kiran Mahajan |
| E-mail(s) |
kiranm@wustl.edu
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| Phone |
3142737728
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| Organization name |
Washington University
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| Department |
Surgery
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| Lab |
Kiran Mahajan Lab
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| Street address |
CB 8242 600 South Euclid Ave
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| City |
St. Louis |
| State/province |
Missouri |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL21290 |
| Series (2) |
| GSE169133 |
Targeted Escalation of an Acetylated HOXB13-dependent Tyrosine Kinase Super-Enhancer Network Promotes Prostate Tumor Autonomy |
| GSE169134 |
Targeted Escalation of Acetylated HOXB13 Regulated Super-Enhancers Promotes Prostate Tumor Autonomy |
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| Relations |
| BioSample |
SAMN18344075 |
| SRA |
SRX10372586 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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