Additional file 2 of guidedNOMe-seq quantifies chromatin states at single allele resolution for hundreds of custom regions in parallel
Creators: name: Schwaiger, Michaela
name: Mohn, Fabio
name: Bühler, Marc
name: Kaaij, Lucas J. T.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified

description: Supplementary Material 2: Figure S1. Benchmarking guidedNOMe-seq. (A) Scatter plot comparing fragment number over ROI between replicates of guided NOMe-seq libraries (B) Scatter plot comparing fragment number over ROI before and after UMI correction (C) Scatter plot comparing fragment number over ROI between replicates of two array capture NOMe-seq libraries (D) The percent of mapped reads that cover the assay specific target regions between 3 different target enrichment approaches. Array capture data from Sönmezer et al (E)Density plot showing the percentage of informative (spanning the three chromatin state quantification windows) reads when performing Array capture and guidedNOMe-seq. (F) Line graph comparing oligo synthesis prices when performing either array capture NOMe or guidedNOMe targeting different numbers of loci, as indicated. Figure S2. Basic NOMe-seq analysis. (A) Scatter plot showing the sgRNA on target scores predicted by 6 different algorithms, as indicated versus the observed associated ROI read counts Violin plots showing the sgRNA on target scores predicted by. (B) Line graphs showing the running mean smoothed levels of simple repeats and low complexity regions in the 1500 ROI ordered by the observed fragment counts per ROI. (C) Genome browser view of a ROI showing from top to bottom: (top) the position of the 4 sgRNAs designed up and downstream of the ROI, (middle) guidedNOMe-seq read coverage of the intended ROI flanked by the sgRNAs (left) and background reads originating most likely from Cas9 cutting at the 5’ end and DNA breakage at the 3’ end (right) (D) Barplot showing presence of off target reads that can be linked to predicted off target sgRNA target sites. Individual bars show read linkage split on sgRNA off targets with and without PAM sequence and random controls, as indicated. (E) Shows correlation plots of two wild type replicates depicting the average observed GpC protection per position between the 4x10^6 reads and all other subsampled amounts, as indicated. ROI TF relationship is highlighted by different colors, as indicated (F) Scatterplots showing the reproducibility between replicates of the TF chromatin state classification. Every ROI is depicted as a single dot. We subsampled the reads, as indicated above the average plot. ROI with two or more informative fragments are shown. Figure S3. Quantification of chromatin state changes using guidedNOMe-seq upon ChAHP perturbation. (A) guidedNOMe-seq example of a CTCF binding site and its surrounding (~250bp +/-). Top plot shows the average protection value of the GpCs at the indicated location. Bottom plot shows protected and unprotected state of the GpCs in individual DNA molecules. DNA molecules are sorted based on the chromatin state they are in, as indicated in the barplot on the right. Single ROI plots were generated under wild type conditions and after 24H Ctcf depletion, as indicated. Putative nucleosome protected regions, here assumed to results in stretches of DNA larger then &gt;100bp protected from methylation by M.CviPI have been manually highlighted in grey (B) guidedNOMe-seq example of a CTCF-Adnp co-bound ROI and its surrounding (~250bp +/-). Top plot shows the average protection value of the GpCs at the indicated location. Bottom plot shows protected and unprotected state of the GpCs in individual DNA molecules. DNA molecules are sorted based on the chromatin state they are in, as indicated in the barplot on the right. Single ROI plots were generated under wild type and Adnp-/- conditions, as indicated. (C) Scatterplot showing changes in chromatin state between wild type and Adnp-/- conditions. ROI are colored on TF (REST, CTCF no ADNP and CTCF with ADNP), see right side of the plots. Every symbol is a ROI and the x-axis and y-axis show the fraction of DNA molecules under wild type and Adnp-/- conditions that is in the chromatin state indicated on top of the plot. edgeR was used to identify ROI that significantly change between wild type and Adnp-/- conditions and the shape of the individual ROI corresponds to the edgeR output as indicated on the right hand site of the plots. (D) Scatterplot showing changes in chromatin state between wild type and Ctcf depleted conditions. ROI are colored on TF (REST, CTCF no ADNP and CTCF with ADNP), see right side of the plots. Every symbol is a ROI and the x-axis and y-axis show the fraction of DNA molecules under wild type and Ctcf depleted conditions that is in the chromatin state indicated on top of the plot. edgeR was used to identify ROI that significantly change between wild type and Ctcf depleted conditions and the shape of the individual ROI corresponds to the edgeR output as indicated on the right hand site of the plots.  (E) Stacked barplot showing the three different ROI groups and the amount and direction of significantly changed chromatin states as determined by edgeR between the wild type and Adnp-/-.