Targeted enrichment of the MHC region of tumor cell lines. (IMAGE)
Caption
(A) A detailed diagram illustrating the CRISPR-based targeted haplotype-resolved assembly of the MHC region. (B) The positions of two sets of sgRNAs targeting the MHC regions. The lines and boxes colored with semitransparent blue and red represent two sets of sgRNAs showing the targeting sites, respectively. The targeted region spans from 28.6 Mb to 33.4 Mb, while the violet, blue, and green colored boxes represent the regions encompassing HLA class I, class III, and class II genes, respectively. The amber boxes denote the genes used for quantitative real-time PCR analyses, listed according to their coordinates on the GRCh38 reference. (C) The relative enrichment of the targeted MHC region of the tumor cell lines was validated using quantitative real-time PCR. The fold enrichment was quantified relative to the HFE gene, located upstream of the targeted region, and normalized to cells treated without sgRNAs. The relative enrichment for RNF8, a gene outside the targeted MHC region, was tested as a negative control. Data were obtained from two independent experiments. (D) The sequencing coverage of the targeted MHC region of the A549 cell line based on data from the 10x Genomic linked-read and PacBio HiFi sequencing platforms. The beige area, encompassed by the two sets of sgRNAs (red and blue bars at the top), highlights the targeted MHC region. The dashed lines indicate a 50-fold enrichment for 10x Genomics linked-read data (green) and a 30-fold enrichment for PacBio HiFi data (orange).
Credit
Haozhe Yuan, Mengping Jiang, Xingyu Xu, Jialiang Zhu, Shulong Dong, Weida Meng, Dandan Zhang, Jiakang Ma, Yicheng Lin, Ziqiang Chen, Shaoyang Sun, Wenqing Qiu, Yun Liu
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