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| Status |
Public on May 23, 2016 |
| Title |
P300 ChIP (16 week) |
| Sample type |
SRA |
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| Source name |
Human kidney ChIP P300-16wk
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| Organism |
Homo sapiens |
| Characteristics |
genotype: wild type
tissue: renal cortex
age: 16 weeks 2 days
Stage: Fetal 17wk
Sex: unknown
pooled sample: no
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| Treatment protocol |
For ChIP-seq from 16-17 week human fetal kidneys, specimens were immediately incubated in 1% PFA crosslink buffer (PMID:22902740) and stored on ice for a maximum of 30 minutes. Samples were then microdissected to remove the cortex (~0.5 mm) and incubated for an additional 30 minutes at room temperature in fresh 1% PFA crosslink buffer. Crosslinking was stopped by the addition of glycine. Tissue was washed with PBS containing protease inhibitors (PI) and resuspended in fresh PBS+PI. Tissue was spread across 1.5 mL Eppendorf tubes and homogenized with a motorized pestle. Homogenized tissue was pelleted, resuspended in mouse ChIP lysis buffer (PMID:22902740), and recombined. The tissue was lysed with the aid of a B dounce homogenizer and incubated on ice for 20 minutes. Processing of the samples from this point was carried forward using the mouse ChIP protocol (PMID:22902740). Sequencing libraries were made using the ThruPLEX-FD Prep Kit (Rubicon Genomics). Libraries were submitted to the USC Epigenome Center for sequencing on the Illumina HiSeq 2000.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Human fetal kidney tissue was microdissected to remove the cortex and incubated for 30 minutes at room temperature in crosslink buffer. Crosslinking was stopped by the addition of glycine. Tissue was washed with PBS containing protease inhibitors, homogenized, pelleted, and lysed in ChIP lysis buffer with the aid of a B dounce homogenizer. Processing of the samples from this point was carried forward using the mouse ChIP protocol (see Park et al, 2012; PMID: 22902740 for experimental conditions and buffer components).
Sequencing libraries were made using the ThruPLEX-FD Prep Kit (Rubicon Genomics) according to manufacturer’s instructions. In brief 10-50ng of DNA was end repaired, A-tailed and ligated with adapters, amplified with indexing reagents, and DNA libraries purified using Ampure magnetic beads. Libraries were submitted to the USC Epigenome Center for sequencing on the Illumina HiSeq 2000.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
genome build: hg19
ChIP Antibody: p300 Antibody (C-20) (Santa Cruz, cat# sc-585)
URL: http://www.gudmap.org/gudmap/pages/ngd_submission.html?id=GUDMAP%3A22350
GUDMAP ID: 22350
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| Data processing |
Mapped ChIP-seq and input data were analyzed using QuEST 2.4 software (Valouev et al., 2008; PMID: 19160518). QuEST peak calling parameters: We used a “transcription factor” setting (for SIX1 and SIX2); bandwidth of 30 bp, regions size of 300 bp) or a ‘histone’ setting (H3K27ac, P300); bandwidth of 100 bp, regions size of 1000 bp). Peak calling stringency was specified as following: a sample-dependent ChIP enrichment fold, 3-fold ChIP over input enrichment were used to seed the regions, and 3-fold ChIP enrichment was assigned for extending the regions. FDR for detecting the bound regions was evaluated by allocating the same number of mapped reads from a separate input library and performing QuEST analysis using the same parameters.
All ChIP-seq sequences were mapped to hg19 using Novoalign software (Novocraft). Novoalign alignment parameters: Single-end reads trimming of 10 bp, polyclonal read filter: 7,10 0.4,2, maximum alignment score acceptable: 120.
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| Submission date |
Dec 11, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
GUDMAP Developers |
| E-mail(s) |
gudmap-db@gudmap.org
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| Phone |
+44 131 651 8500
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| Organization name |
IGMM MRC Human Genetics Unit
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| Lab |
GUDMAP Database Group
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| Street address |
Crewe Road
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| City |
Edinburgh |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE75948 |
Genome-wide maps of SIX1, SIX2, and active loci in human fetal kidneys generated from ChIP-seq data. |
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| Relations |
| BioSample |
SAMN04332460 |
| SRA |
SRX1478537 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1970524_22350_P300_ACAGTG.bed.gz |
863.2 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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