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| Status |
Public on Mar 07, 2018 |
| Title |
11-10T.H4K16ac.Old |
| Sample type |
SRA |
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| Source name |
lateral temporal lobe
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| Organism |
Homo sapiens |
| Characteristics |
study group: Old
age (years): 63
antibody: Millipore, #07-329
tissue: lateral temporal lobe
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| Growth protocol |
Frozen brain tissue samples from lateral temporal lobe (Brodmann area 21 or 20) were obtained from the Center of Neurodegenerative Disease Research (CNDR) biobank at the University of Pennsylvania in accordance with Institutional Review Board approved protocols. A neuropathological diagnosis of AD was established based on the presence of plaques and tangles using the CERAD scores and Braak stages, respectively. All selected AD cases had high level of AD neuropathological changes (Braak=V/VI and CERAD=C). The Young and Old control brains had no or minimal neuritic amyloid plaques (CERAD=0) or neurofibrillary tangles (CERAD=0). None of the AD cases had other coincident neurodegenerative diseases. Control subjects had no deposits consistent with a frontotemporal lobar degeneration or Lewy body related pathology diagnosis. AD cases with severe neuronal loss were not included. The neuronal loss was originally assessed through semi-quantitative measurements by hematoxylin and eosin (H&E) staining by board-certified neuropathologists of the CNDR. The H&E scoring for neuronal loss ranges from 0-3 where 0 signifies no neuronal loss and 3 is severe neuronal loss. Only cases with neuronal loss of 1 or 2 (mild or moderate) were included.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, 200 mg brain tissue from each patient was minced on ice and nuclei were prepared by dounce homogenization in nuclei isolation buffer (50 mM Tris-HCl at pH 7.5, 25 mM KCl, 5 mM MgCl2, 0.25 M sucrose) with freshly added protease inhibitors and sodium butyrate, followed by ultracentrifugation on a 1.8 M sucrose cushion. Nuclei pellet was resuspended in 2 ml PBS and crosslinked in 1% formaldehyde for 10 min at room temperature. Crosslinking reactions were quenched with addition of glycine to 125 mM for 5 min followed by two washes in cold PBS. 2 x 106 nuclei were lysed in nuclei lysis buffer (10 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine) with freshly added protease inhibitors and sodium butyrate and chromatin was sheared using a Covaris S220 sonicator to ~250 bp size. Equal aliquots of sonicated chromatin were used per immunoprecipitation reaction with 5 ul H4K16ac antibody (Millipore, #07-329) preconjugated to Protein G Dynabeads (Life Technologies), and 10% of the amount was saved as input. ChIP reactions were incubated overnight at 4°C with rotation and washed three times in wash buffer.
Immunoprecipitated DNA was eluted from the washed beads, purified and used to construct sequencing libraries with 5 ng of DNA (ChIP or Input) using the NEBNext Ultra DNA library prep kit for Illumina (New England Biolabs, NEB). Libraries were multiplexed using NEBNext Multiplex Oligos for Illumina (dual index primers) and single-ended sequenced (75 bp) on the NextSeq 500 platform (Illumina) in accordance with the manufacturer’s protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
processed data file: H4K16ac-Input.Old.bw
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| Data processing |
Alignment: ChIP-seq tags generated with the NextSeq 500 platform were demultiplexed with the bcl2fastq utility and aligned to the human reference genome (assembly NCBI37/hg19) using Bowtie v1.1.1 allowing up to two mismatches per sequencing tag (parameters -m 1 --best).
PCR deduplication: Aligned tags were filtered so that when N tags aligned to the same locus, only one representative tag was kept.
Tracks: Generation and visualization of ChIP-seq tracks was conducted as follows. BED files of each pooled study group dataset were converted into coverage maps using the BEDtools utility genomeCoverageBed. Resulting bedGraphs were scaled by using the RPM (reads per million) coefficient, a measure of the millions of tags sequenced per sample to correct for sequencing efficiency biases, and subsequently normalized by subtracting an Input coverage map. Finally, BigWig files were generated and uploaded on the UCSC (University of California Santa Cruz) Genome Browser.
Differential peaks were assessed in the following way. Peaks were first detected using MACS2 (tag size = 75 bp; FDR < 1x10-5) on H4K16ac and input from patient samples pooled over study group. Within each study group, peaks with termini within 150 bp were merged. The MTL method (Chen, X., et al., Cell, 2008. 133(6): p. 1106-17) was then used to compare H4K16ac enrichment across the three study groups. A region of analysis across the three study groups was defined by having at least one peak called in Young, Old or AD. Furthermore, if peaks across the three study groups had their centers within 200 bp distance, the entire area including these peaks (from peak to peak termini) was considered one unique region of analysis. Per-patient H4K16ac enrichment scores were calculated by summing H4K16ac tags overlapping this unique region of analysis and adjusting them by a reads-per-million (RPM) scalar coefficient and by the size of the region of analysis, then subtracting input. AUC values were then transformed in log2(AUC + 1) and statistical significance of differential H4K16ac enrichments was assessed by performing a standard Student's t-test for a pairwise comparisons (i.e., Young vs Old) with alpha 0.05.
Genome_build: hg19
Supplementary_files_format_and_content: BigWig files are created by method described in "Tracks" above.
Supplementary_files_format_and_content: Bed files: ChIP-seq peaks with differential enrichment between the experimental conditions.
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| Submission date |
Oct 06, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Donahue |
| Organization name |
The University of Pennsylvania
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| Department |
Cell & Developmental Biology
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| Lab |
Zaret Lab
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| Street address |
3400 Civic Center Blvd, Bldg 421
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE84618 |
Dysregulation of the epigenetic landscape of normal aging in Alzheimer's disease [ChIP-Seq] |
| GSE104705 |
Dysregulation of the epigenetic landscape of normal aging in Alzheimer's disease |
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| Relations |
| BioSample |
SAMN07757287 |
| SRA |
SRX3257634 |
| 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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