title: GSM3324486: Human10_ChIP_Pol2_ZIKVpositive_24h; Homo sapiens; ChIP-Seq
library_construction_protocol: For RNA-seq acrophages were washed once with 1XPBS, gently scraped off in 1XPBS, pelleted at 4C and stained with zombie violet viability dye [BioLegend] in the dark. Cells were washed once with FACS buffer (1X PBS, 3% fetal bovine serum, and 1 mM EDTA) and fixed and permeabilized with 4% PFA [Electron Microscopy Sciences, 15710-S], 0.1% saponin [Sigma-Aldrich, 47036] in molecular grade PBS supplemented with 1:100 RNasin Plus RNase Inhibitor [Promega, N2615] for 30' at 4C. Cells were washed in Wash Buffer: 1XPBS containing 0.2% BSA, 0.1% saponin, and 1:100 RNasin Plus RNase Inhibitor and blocked for 10' with Human Fc Block (1:500) [BioLegend] in Staining Buffer: 1XPBS containing 1%BSA, 0.1% saponin, and RNasin Plus RNase Inhibitor. HMDMs were then stained with 4G2 antibody [BioXcell] conjugated to AF647 [Thermo Scientific Cat#A20186] for 30' at 4C, washed twice, resuspended at 5-10E6 cells/ml in Sort Buffer (PBS containing 0.5% BSA, and 1:25 RNasin Plus RNase Inhibitor) and sorted into ZIKV+ and ZIKV- cells on the FACSAria [BD Biosciences] at La Jolla Institute for Allergy for Immunology. Gates were set with reference to negative controls. After sorting, cells were pelleted at at 4C, supernatant discarded and total RNA isolated using Ambion's RecoverAll Total Nucleic Acid Isolation Kit [AM1975], starting at the protease digestion step. All steps were performed per manufacturer recommendations with the following modification. Cells were incubated in digestion buffer for 3 hours at 50C supplemented with RNasin Plus RNase Inhibitor. RNA was treated with in column DNase per manufacturer instructions, eluted and RNA quality determined by BioAnalyzer using the Eukaryote Total RNA Pico Chip. Samples with RIN values greater than 8.0 were used for library preparation. For ChIP-seq macrophages were cross-linked with 1% formaldehyde for 15 minutes in the presence of 1 mM sodium butyrate and quenched with 0.125M glycine. Preparation of HMDMs for FACS was performed as described above for RNA-seq except that 1x cOmplete protease inhibitors (Roche) and 1 mM sodium butyrate [Sigma] were included in all buffers instead of RNasin Plus RNase Inhibitor. Following FACS cells were washed, pelleted and snap-frozen. Strand-specific total RNA-seq libraries from ribosomal RNA-depleted were prepared using the TruSeq kit (Illumina) according to the supplied protocol. ChIP-seq was essentially performed as described (Heinz et al., 2013), except that libraries were prepared using NEB Ultra II DNA library prep kit reagents.
attributes.tag: GEO Accession
attributes.value: GSM3324486