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. 2021 Aug 16;22(16):8792. doi: 10.3390/ijms22168792

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Chromatin status of the functional SNP rs684232 in 22Rv1 cells. (A) Reporter gene expression level for rs684232 SNP region in the DiR-seq analysis in 22Rv1 cells. T allele showed increased enhancer activity relative to the C allele. Mean ± SEM of three independent experiments. *** p < 0.001, two-tailed Student’s t-test. (B) Reporter gene expression level for rs684232 SNP region in the DiR-qPCR assay in 22Rv1 cells. Mean ± SEM of three independent experiments. ** p < 0.01, two-tailed Student’s t-test. (C) Normalized luciferase activity for the rs684232 region in 22Rv1 cells. T allele showed increased enhancer activity relative to the C allele. Mean ± SEM of three independent experiments. * p < 0.05, two-tailed Student’s t-test. (D) Sanger sequencing chromatography of FAIRE DNA for the rs684232 site in 22Rv1. The position of rs684232 is highlighted in a yellow square. (E) Allele-specific enrichment of rs684232 site in FAIRE DNA determined in 22Rv1 cells by AS-qPCR. Mean ± SEM of three independent experiments. *** p < 0.001, two-tailed Student’s t-test. (F) Fold enrichment of the rs684232 region in H3K27ac and H3K4me3 ChIP DNA in 22Rv1. Nonspecific immunoglobulin G (IgG) and Input as the negative control. Mean ± SD of three technical replicates. *** p < 0.001, two-tailed Student’s t-test. (G) Sanger sequencing chromatography of rs684232 in H3K27ac and H3K4me3 ChIP DNA in 22Rv1 cells. IgG and Input as the negative control. The position of rs684232 is highlighted in a yellow square. (H) Allele-specific enrichment of rs684232 region in ChIP DNA of H3K27ac and H3K4me3 modification determined by AS-qPCR in 22Rv1. Mean ± SD of three technical replicates. * p < 0.05, *** p < 0.001, two-tailed Student’s t-test. (I) Chromatin open status analysis of the rs684232 region in the two genome-edited cell lines 22Rv1(−/−) #1 and 22Rv1(−/−) #2. Mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, two-tailed Student’s t-test. (J) Sanger sequencing chromatography of the FAIRE DNA around rs684232 sites in the two genome-edited 22Rv1 cell lines.

Chromatin status of the functional SNP rs684232 in 22Rv1 cells. (A) Reporter gene expression level for rs684232 SNP region in the DiR-seq analysis in 22Rv1 cells. T allele showed increased enhancer activity relative to the C allele. Mean ± SEM of three independent experiments. *** p < 0.001, two-tailed Student’s t-test. (B) Reporter gene expression level for rs684232 SNP region in the DiR-qPCR assay in 22Rv1 cells. Mean ± SEM of three independent experiments. ** p < 0.01, two-tailed Student’s t-test. (C) Normalized luciferase activity for the rs684232 region in 22Rv1 cells. T allele showed increased enhancer activity relative to the C allele. Mean ± SEM of three independent experiments. * p < 0.05, two-tailed Student’s t-test. (D) Sanger sequencing chromatography of FAIRE DNA for the rs684232 site in 22Rv1. The position of rs684232 is highlighted in a yellow square. (E) Allele-specific enrichment of rs684232 site in FAIRE DNA determined in 22Rv1 cells by AS-qPCR. Mean ± SEM of three independent experiments. *** p < 0.001, two-tailed Student’s t-test. (F) Fold enrichment of the rs684232 region in H3K27ac and H3K4me3 ChIP DNA in 22Rv1. Nonspecific immunoglobulin G (IgG) and Input as the negative control. Mean ± SD of three technical replicates. *** p < 0.001, two-tailed Student’s t-test. (G) Sanger sequencing chromatography of rs684232 in H3K27ac and H3K4me3 ChIP DNA in 22Rv1 cells. IgG and Input as the negative control. The position of rs684232 is highlighted in a yellow square. (H) Allele-specific enrichment of rs684232 region in ChIP DNA of H3K27ac and H3K4me3 modification determined by AS-qPCR in 22Rv1. Mean ± SD of three technical replicates. * p < 0.05, *** p < 0.001, two-tailed Student’s t-test. (I) Chromatin open status analysis of the rs684232 region in the two genome-edited cell lines 22Rv1(−/−) #1 and 22Rv1(−/−) #2. Mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, two-tailed Student’s t-test. (J) Sanger sequencing chromatography of the FAIRE DNA around rs684232 sites in the two genome-edited 22Rv1 cell lines.