DiagenodeのiDealChIP-seq Kit for Transcription Factorsは、ChIPとその後に行うリアルタイムPCRによってオンターゲットな領域のdCas9を特異的に濃縮することで、ゲノム内のdCas9結合を調査する堅牢なワークフローが特徴です。さらに、9dCas抗体で免疫沈降したDNAのシーケンス解析は、エフェクターに融合したdCas9の正しい結合を確認するために不可欠であるゲノムワイドなオフターゲット効果の可能性を示しています。
ChIP was performed on sheared chromatin from 4,000,000 HEK293T cells using the iDeal ChIP-seq Kit for Transcription Factors, 5 µl of the polyclonal Cas9 antibody and 1 µg of the negative control IgG. Primers specific for the human H19, GAPDH, Myt1 and SLC17A4 were used for the qPCR. The figure shows the recovery, expressed as a percent of input (the relative amount of immunoprecipitated DNA compared to input after qPCR analysis).
ChIP was performed on sheared chromatin from 4 000 000 HEK293T cells using the iDeal ChIP-seq kit for Transcription Factors, 5 µl of the polyclonal Cas9 antibody and 1 µg of the negative control IgG. The libraries were sequenced on Illumina HiSeq 3000 in 1X50 pb. The picture shows the read distribution for the manual IP (up), automated IP (middle) and IgG (bottom) samples. A-B. Peaks distribution of the three datasets in the region surrounding H19 (A) and a representative region of the genome (B) for HEK293T cells expressing a sgRNA for H19. C-D. Peaks distribution of the three datasets in the region surrounding GAPDH (C) and YIPF4 (D) for HEK293T cells expressing a sgRNA for GAPDH
2. Confirmation of binding specificity of sgRNA and detection of off-target bindings by ChIP-seq
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