GSM9350834: HSV1 A3A overexpressed HEK293 input; Homo sapiens; ChIP-Seq
Twenty million HSV-1 infected or mock control cells were grown to near-confluence, followed by crosslinking them with 1% formaldehyde for 10 minutes and quenching with glycine. After washing and lysing, the cells were stored until the chromatin could be prepared. Chromatin was then sheared by sonication to achieve the desired fragment size of 200–500 bp, with 2% of the fragmented material set aside as input DNA. Chromatin Immunoprecipitation (ChIP) was performed using 8 µg of specific polyclonal antibodies: anti-APOBEC3A and anti-APOBEC3G for Jurkat cells, anti-HA for RPE-1 cells, or anti-tGFP for HEK293-AD cells. Concurrently, DNA:RNA Hybrid Immunoprecipitation (DRIP) was conducted on the same samples using the S9.6 monoclonal antibody. For DRIP, 6 µg of genomic DNA was first isolated (omitting RNase A), fragmented by sonication, and then incubated overnight with S9.6 antibody-coated Protein G magnetic beads; a separate sample was treated with RNase H as a control. Following washing steps, the immunoprecipitated material was eluted using proteinase K, and the final DNA was purified using Ampure XP beads and quantified. Illumina TruSeq ChIP Sample Preparation protocol were used to DRIP-seq. DRIP DNA were end-repaired and indexed adapters were ligated to the inserts. Purified ligation products were then amplified by PCR. Amplified libraries were sequenced (at the EMBL Genomics Core Facility, Heidelberg) using an Illumina HiSeq 2500 instrument in the 2x125 paired-end sequencing mode, and Illumina NovaSeq X Plus PE150 (at Novogene Ltd.).