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| Status |
Public on Feb 15, 2019 |
| Title |
hESC_53BP1-Novus replicate 1 |
| Sample type |
SRA |
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| Source name |
Cell Line
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Human Embryonic Stem cell H9
cell line: H9
genotype/variation: wildtype
chip antibody: 53BP1(Novus,NB100-304)
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| Treatment protocol |
Neuronal maturation assay was performed using STEMdiff™ neuron differentiation kit (STEMCELL Technologies, #08500) and STEMdiff™ neuron maturation kit (STEMCELL Technologies, #08510). ES cells were seeded onto AggreWell800 plates (STEMCELL Technologies, #34811) and fed with neural induction medium (STEMCELL Technologies, #05835) to form uni-sized embryoid bodies (EBs). On day 5, EBs were re-plated onto Matrigel-treated 6-well plates. Neural differentiation started from day 11 to day 16. On day 17, cells were treated with Accutase® (STEMCELL Technologies) and seeded and fed with neuronal maturation medium till days 22–24. RNA samples were collected on days 10, 11, and 22 for deep-sequencing analysis. Cells were also seeded onto multi-chamber slides for immunofluorescence (IF) analysis.
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| Growth protocol |
H9/WA09 cells (WiCell) were grown on Matrigel with reduced growth factors (ThermoFisher Scientific, #354230) in mTeSR1 medium (STEMCELL Technologies, #85850) at 37°C and 5% CO2. To induce neural lineage, cells were plated in PSC neural induction medium (Life Technologies) at 15–25% confluency, with medium changed every other day. On day 7, cells were harvested or expanded for further analyses.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 2 mg of nuclear extracts was incubated with primary antibody overnight at 4°C. Extract and antibody were added to protein A and protein G Dynabeads™ (ThermoFisher Scientific, #10002D and 10004D) for 4 h at 4°C, washed with PBST, and eluted with 0.1 M glycine (pH 2.3). Eluates were neutralized with 1.5 M Tris buffer (pH 8.8).
Cells were harvested in PBS. Cytoplasmic fractions were extracted using buffer A with 1× protease inhibitors and 1 mM DTT. Nuclear pellets were cross-linked by 1.1% formaldehyde in buffer B with 1× protease inhibitors and 1 mM DTT; washed; and lysed in lysis buffer 3 with 1× protease inhibitors, 1 mM DTT, and 1 mM PMSF. The fixed and lysed nuclear extract was sonicated with Bioruptor® Pico (Diagenode) 10 times for 15 s each , with 45-s intervals. Chromatin was added to Dynabeads™ (Life Technologies) prebound with 4 µg of antibodies. For UTX chromatin immunoprecipitation (ChIP), chromatin and antibodies were incubated overnight, followed by 4 h of recapture with Dynabeads™ the next day. After incubation, beads were washed and immunoprecipitates were eluted. DNA from eluates was recovered by the GeneJET FFPE DNA purification kit (ThermoFisher Scientific, #K0882). ChIP-qPCR signals were calculated as the percentage of input. DNA libraries were generated using the NEBNext™ Ultra DNA Library Prep kit (NEB, #E7370S) and sequenced at the St. Jude Hartwell Center.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
processed data file:
hESC_53BP1-Novus.merge.broadPeak
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| Data processing |
Basecalls performed using CASAVA
Single-end reads of 50 bp were obtained. BWA (version 0.5.9-r26-dev, default parameter) was used to align reads to human genome hg19. Duplicated reads were marked by Picard (version 1.65(1160)), and uniquely mapped reads were retained by SAMtools (parameter “-q 1 -F 1024” version 0.1.18 (r982:295)). When clear peaks were observed by inspection on Integrated Genomics Viewer (Broad Institute) and there was high consistency among replicates, peaks for each replicate were called by MACS2 (version 2.0.9 20111102). Peaks within 100 bp were merged by bedtools (version 2.17.0).
Almost every CHIPSEQ sample have 20M unique mapped reads, only few have less but more than 15M unique mapped reads. We confirmed quality control results were good following the ENCODE guideline for CHIPSEQ, reads were then extend to the estimated fragment size and converted to bigwig files. With clear peaks observed by inspection on Integrated Genomics Viewer (Broad Institute) and there was high consistency among replicates. We further extend reads to the estimated fragment size and counted at each called peaks, pearson correlation coefficients were calculated among replicates suggested high reproducibility.
Genome_build: hg19(GRCh37)
Supplementary_files_format_and_content: broadpeak or txt file with peaks
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| Submission date |
Dec 14, 2017 |
| Last update date |
Feb 15, 2019 |
| Contact name |
Beisi Xu |
| E-mail(s) |
beisi.xu@stjude.org
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| Organization name |
St Jude Children's Research Hosipital
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| Department |
Center for Applied Bioinformatics
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| Street address |
262 Danny Thomas Pl
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| City |
Memphis |
| State/province |
Tennessee |
| ZIP/Postal code |
38105 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE108114 |
UTX and 53BP1 co-regulate genetic programs for neural differentiation of human embryonic stem cells [ChIP-seq] |
| GSE108116 |
UTX and 53BP1 co-regulate genetic programs for neural differentiation of human embryonic stem cells |
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| Relations |
| BioSample |
SAMN08178717 |
| SRA |
SRX3472377 |
| 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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