title: GSM2526069: 404_Sirt1_i73; Macaca mulatta; ChIP-Seq
library_construction_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.