100% HCV infected (generated as described above) and non-infected Huh7.5 cells were fixed with 1% formaldehyde to cross-link proteins to DNA. Cells were then lysed, and chromatin was fragmented by sonication (Covaris S220) to generate DNA fragments of 200–1000 bp. ChIP was carried out using 5 μg of either anti-RAD21 Ab (BETHYL) or 5 μg of specific antibody against H3K9Ac Abs (Milipore) or normal Rabbit IgG Ab as control (Milipore) and Magna ChIP G kit (Milipore). Cross-links were reversed, and DNA purified. The purified DNA was then used for RT- PCR and next generation sequencing (NGS) using Illumina HiSeq 2500.
Normal rabbit igg ab
Manufactured by Merck Group
Normal Rabbit IgG Ab is a laboratory reagent used as a control antibody in various immunoassays and immunochemistry applications. It is derived from the serum of healthy rabbits and functions as a representative non-specific immunoglobulin G (IgG) antibody.
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2 protocols using normal rabbit igg ab
1
ChIP-Seq Analysis of HCV Infection
2
ChIP-seq protocol for histone modifications
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ChIP assays were performed using the Magna Chip G kit (Milipore) according to the manufacturer’s instructions. 100% HCV infected (generated as described above) and non-infected Huh7.5 cells were fixed with 1% formaldehyde to cross-link proteins to DNA. Cells were then lysed, and chromatin was fragmented by sonication (Covaris S220) to generate DNA fragments of 200–1000 bp. Chromatin was incubated overnight with either specific antibodies against H3K9Ac, H3K43Me, H3K93Me and H3K273Me (Millipore) or normal Rabbit IgG Ab (Milipore) as a negative control for the immunoprecipitation experiments. Cross-links were reversed, and DNA purified.
Libraries for next generation sequencing (NGS) were prepared by using NEBNext Ultra TM DNA Library Prep Kit for Illumina (NEB) and sequenced using Illumina HiSeq 2500 platform with single-end reads of 50 bp according to the manufacturer’s instructions. For each ChIP, at least three biological replicates were performed. For resected liver tissues: ChIP assays were performed using the iDeal ChIP seq kit for Histones (Diagenode) according to the manufacturer’s instructions. For each sample, 20–40 mg of liver tissue was chopped, homogenized, and subjected to ChIP using H3K9Ac or a Rabbit IgG Ab as negative control (Diagenode).
Libraries for next generation sequencing (NGS) were prepared by using NEBNext Ultra TM DNA Library Prep Kit for Illumina (NEB) and sequenced using Illumina HiSeq 2500 platform with single-end reads of 50 bp according to the manufacturer’s instructions. For each ChIP, at least three biological replicates were performed. For resected liver tissues: ChIP assays were performed using the iDeal ChIP seq kit for Histones (Diagenode) according to the manufacturer’s instructions. For each sample, 20–40 mg of liver tissue was chopped, homogenized, and subjected to ChIP using H3K9Ac or a Rabbit IgG Ab as negative control (Diagenode).
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