Rabbit non immune serum igg

ChIP assay was performed using protocol from Dr. Farnham’s laboratory (https://bohdan-khomtchouk.github.io/docs/Chromatin-Immunoprecipitation-ChIPs-Protocol-from-Farnham-Lab.pdf). Chromatin DNA prepared from 1 × 10⁷ TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R cells was used for ChIP assays using anti-KDM4A rabbit polyclonal antibody (25 (link)) and rabbit nonimmune serum IgG (Alpha Diagnostic International). Sequencing libraries were prepared using 10 ng of purified ChIP DNA using Illumina HiSeq Rapid PE Cluster Kit v2 (Illumina, TG-403-2001) according to the manufacturer’s instruction and analyzed by paired-end Illumina HiSeq 2000 sequencing. ChIP-Seq data were aligned to the human reference genome (GRCh38) and HHV-8 reference genome (NC_009333.1) by Partek Flow (Partek).
ChIP DNA was confirmed for successful IP using SYBR Green-based real-time qPCR analysis in the CFX Connect real-time PCR detection system (Bio-Rad). The specific primer set (5′-TTAGAGCGTTTCCAGACCT-3′ and 5′-GAGGATTCAACAGTGATGGGAC-3′) was designed to amplify the potential KDM4A binding site on the IL-10 promoter.

ChIP was performed according to the protocol from Dr. Farnham’s laboratory (http://genomics.ucdavis.edu/farnham). Briefly, chromatin DNA from BCBL-1 cells was harvested after fixation with 1% formaldehyde. Chromatin DNA from 1 x 10⁷ cells was used for each ChIP assay. Anti-JMJD2A rabbit polyclonal antibody, ChIP grade anti-SUMO-2/3 (Abcam, ab81371) mouse monoclonal antibody, ChIP grade anti-H3K9me3 (Abcam, ab8898) rabbit polyclonal antibody and rabbit non-immune serum IgG (Alpha Diagnostic International) were used for the ChIP assays. 50 ng of ChIP’d DNA eluted in 50 μl of ddH₂O was used for ChIP-seq library preparation, according to the protocol from Illumina. DNA fragment libraries (~400 bp) were analyzed for paired-end sequencing on Illumina HiSeq 2000. The ChIP-Seq data was aligned with the KSHV genome and hg19 build by Partek Genomics Suite (Partek Inc. USA). ChIP DNA was confirmed for successful IP using SYBR Green-Based real-time qPCR analysis by CFX connect real-time PCR detection system (Bio-Rad, Richmond, CA). Specific primer sets were designed to amplify potential binding sites. Primer sequences are listed in S2 Table.

ChIP was performed using the protocol from Dr. Farnham’s laboratory (http://genomics.ucdavis.edu/farnham). Briefly, chromatin DNA from control and Dox-treated TREx-F3H3-K-Rta BCBL-1 cells were harvested. Chromatin DNA from 1 x 10⁷ cells was used per antibody for each ChIP assay. ChIP grade anti-SUMO-1 (Abcam, ab32058) and anti-SUMO-2/3 (Abcam, ab3742) specific rabbit polyclonal antibodies, as well as rabbit non-immune serum IgG (Alpha Diagnostic International), were used for the ChIP assays. 50 ng of ChIPed DNA suspended in 30 μl of ddH₂O was used for ChIP-seq library preparation following the protocol from Illumina. Size-selected (400 bp) DNA fragment libraries were used for paired-end high throughput sequencing on Illumina Genome Analyzer_II. The ChIP-Seq data was aligned with the KSHV genome build by Avadis NGS (Strand Scientific Intelligence, San Francisco, CA). Approximately 1~3 x 10⁶ reads were mapped for each sample after filtering and quality control (QC). In this study, we used the target region QC detection method of Avadis NGS to delineate the SUMO-1 and SUMO-2/3 binding patterns. ChIP DNA was verified for successful IP by SYBR Green-Based real-time qPCR using CFX connect real-time PCR detection system (Bio-Rad, Richmond, CA). Specific primer sets were designed around the potential binding sites. Primer sequences were listed in S1 Table.

ChIP assays were performed following the protocol described by Farnham laboratory (provided at http://genomics.ucdavis.edu/farnham). ChIP grade rabbit polyclonal antibodies specific against REST (Millipore, 17–641), H3K4me1 (Abcam, ab8895) and H3ac (Millipore, 06–599), as well as rabbit non-immune serum IgG (Alpha Diagnostic International) were used. ChIP DNA was prepared from 4 × 10⁸ LNCaP cells. ChIP-seq library was prepared following the protocol from Illumina and subjected to sequencing. ChIP-Seq data was aligned onto hg19. The Partek software (Partek Genomics suite 6.6) was used to localize potential REST binding sites. Binding sites were verified by SYBR^® Green Based qPCR using a Bio-Rad CFX96 Real-Time PCR detection system. Specific primer sets were designed around the identified binding sites and listed in Table S1.