Additional file 1 of Multiple distinct domains of human XIST are required to coordinate gene silencing and subsequent heterochromatin formation

Additional file 1: Tables S1–S8. Table S1: Timepoints for FISH and IF-FISH analysis. List of the factors analyzed in the Full XIST 8p HT1080 cell line over the course of multiple days. The factors analyzed either by FISH in the case of Cot-1 or IF in the case of the protein/chromatin are shown in the first column. The second column lists the period of time XIST was induced. The number of cells analyzed, the mean and median z-score calculated as well as the standard deviation (SD) for each condition are listed. The statistical significance of each time point relative to each other were calculated using the Mann–Whitney U test and the p values are listed. Table S2. Summary of Deletion Constructs. Each of the cell lines successfully generated for each type of deletion construct are listed along with the gRNA targeting sequence used to create the deletion and the total number of nucleotides lost from the XIST cDNA sequence. Table S3. Deletion constructs ability to form Cot-1 hole. Probes for Cot-1 were used to measure the formation of a Cot-1 hole by inducible XIST constructs in 8p. The constructs tested are listed in the second column followed by the cell type, number of cells analyzed, the mean and median z-score calculated as well as the standard deviation (SD) for each construct. The statistical significance of each population of deletion constructs’ difference from Full XIST in their z-score s was calculated using the Mann–Whitney U test and the p values are listed. Table S4. The ability of the deletion constructs to remodel chromatin and recruit CIZ1. List of the factors analyzed, cell type, number of cells analyzed, the mean and median z-score calculated as well as the standard deviation (SD) for each construct. The statistical significance of each population of deletion constructs’ difference from Full XIST in their enrichment was calculated using the Mann–Whitney U test and the p values are listed. Table S5. Summary of effect of chemical inhibitors on XIST mediated silencing. The average strength of silencing along with statistical significance of the chemical inhibitor treatments for each allele tested in this paper are shown below. The upper section of the table shows the average strength of silencing and p-values calculated for each condition relative to 5ddox treated Full XIST described in Fig. 3A. The bottom section shows the average strength of silencing and p-values calculated relative to 2ddox treated Full XIST (Fig. 3B). p-values were calculated by t-test. The threshold of statistical significance in this paper was adjusted using the Bonferroni correction. Table S6. Effect of chemical inhibitors on XIST formation of Cot-1 holes. The effect of chemical inhibition for 5 days along with the induction of Full XIST on the formation of Cot-1 holes are shown in this table. The chemical inhibitors and doses used for each test is shown in the first column. The number of cells analyzed, the mean, median and standard deviation of z-score s for each condition are shown in the subsequent columns. The statistical significance of each population of deletion constructs’ difference from induced Full XIST untreated with chemical inhibitors was calculated using the Mann–Whitney U test and the p values are listed. Table S7. Effect of chemical inhibition treatment on XIST chromatin remodelling. The effect of chemical inhibition for 5 days along with the induction of Full XIST on the chromatin remodelling are shown in this table. The chemical inhibitors and doses used for each test is shown in the first column. The number of cells analyzed, the mean, median and standard deviation of z-score s for each condition are shown in the subsequent columns. The statistical significance of each population of deletion constructs’ difference from induced Full XIST untreated with chemical inhibitors was calculated using the Mann–Whitney U test and the p values are listed. Table S8. list of primers used throughout this experiment. The names of the various primers used throughout the experiments described. Primers were labeled either F (forward) or R (reverse) to denote their orientation during amplification. All sequences are orientated 5’ to 3’. Table S9. Table of antibodies used for IF and western blotting.

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