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Sample GSM2526074 Query DataSets for GSM2526074
Status Public on Mar 22, 2017
Title 208_Sirt1_Rh_i87
Sample type SRA
 
Source name Microglia
Organism Macaca mulatta
Characteristics pathology: Uninfected Young
cell type: Brain tissue derived microglial cells
chip antibody: anti-Sirt-1 (Millipore; lot number 2273971; 4ug per ChIP reaction)
Growth protocol Brain immune cells were isolated from all representative areas of the brain (free of meninges), by enzymatic digestion of minced tissue, followed by Percoll (Sigma-Aldrich) gradient.
Extracted molecule genomic DNA
Extraction protocol Cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Lysates were sonicated with a microtip sonicator to shear the DNA. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. For each ChIP reaction, 30 ug of microglia chromatin was precleared with protein A agarose beads (Invitrogen). ChIP reaction was set up using precleared chromatin and Sirt1 antibody (Millipore; lot number 2273971; 4ug per ChIP reaction) and incubated overnight at 4 C. Protein A agarose beads were added and incubation at 4 C was continued for another 3 hr. Immune complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on on Illumina’s HiSeq2500 or NextSeq 500 (50 or 75 nt reads, respectively, single end). Reads were aligned to the Rhesus monkey genome (rheMac2) using the BWA algorithm (default settings). Duplicate reads were removed and only uniquely mapped reads (mapping quality >= 25) were used for further analysis. Alignments were extended in silico at their 3’-ends to a length of 200 bp, which is the average genomic fragment length in the size-selected library, and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWig files. Peak locations were determined using the MACS algorithm (v1.4.2) with a cutoff of p-value = 1e-7.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Description Uninfected Young experment 2
Data processing Sequence Analysis - The 50-nt and 75-nt sequence reads identified using Illumina’s Hi-Seq and NextSeq, respectively, were mapped to the genome using the BWA algorithm with default settings.
The ChIP DNA was processed into an Illumina ChIP-Seq library and sequenced to generate >2 million reads, which were aligned to the M.mulatta genome annotation (MacaM/Oct 2014 assembly) and 6.2 million unique aligns (removed duplicates) were obtained
A signal map showing fragment densities along the genome was visualized in the Integrated Genome Browser (IGB) and MACS peak finding was used to identify peaks. Control data was derived from 5.1 million (positive control) and 5.8 million (negative control) alignments. With default settings, 2141 Sirt-1 meaningful peaks genome-wide were identified.
Genome_build: MacaM/Oct 2014 assembly
Supplementary_files_format_and_content: Displays the fragment density for 32-nt bins along the genome and is used in peak finding using MACS.
 
Submission date Mar 07, 2017
Last update date May 15, 2019
Contact name Maria Cecilia Marcondes
E-mail(s) cmarcondes@SDBRI.org
Phone 858-200-7197
Organization name San Diego Biomedical Research Institute
Street address 10865 Road to the Cure, Suite 100
City San Diego
State/province CA
ZIP/Postal code 92121
Country USA
 
Platform ID GPL21120
Series (1)
GSE95793 Sirtuin 1-chromatin-binding dynamics point to a common mechanism regulating microglial inflammatory targets in SIV infection and in the aging brain
Relations
BioSample SAMN06546082
SRA SRX2618706

Supplementary file Size Download File type/resource
GSM2526074_5_2910Scripps_s5_208_Sirt1_Rh_i87_uniqnorm_signal.bw 120.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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