7SK ChIRP-seq is specifically RNA dependent and conserved between mice and humans.

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Reginald Montgomery
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1 Supplementary Figure 1 7SK ChIRP-seq is specifically RNA dependent and conserved between mice and humans. Regions targeted by the Even and Odd ChIRP probes mapped to a secondary structure model 56 of the 7SK snrna. The Even and Odd probes each independently cover the full length of the RNA. b, qrt-pcr analysis of RNA enriched from mouse ES cell 7SK ChIRP-seq. Chromatin samples were first treated with (+RNase) or without (-RNase) and recovery of 7SK snrna, 18S rrna, U1 snrna, Malat1, Neat1, β-actin, Gapdh, snora16a, snora44, and snord99 were assessed. Data are mean and s.d. of biological duplicate experiments.

2 c, Scaled metagene analysis of 7SK ChIRP-seq (red) and Pol II ChIP-seq (black) enrichment at actively transcribed mouse ES genes. d, 7SK ChIRP-qPCR analysis from mouse ES cell. Data are mean and s.d. of biological duplicate experiments. e, 7SK ChIRP-seq reads mapped to the 13kb transcribed region of the mouse ribosomal DNA (rdna) locus (GeneBank ID: U ). The 18S (red) 5.8S (green) and 28S (blue) regions are highlighted. The percentage of reads mapping to the rdna locus is shown for the Odd, Even, and Input experiments. f, qrt-pcr analysis of RNA enriched from human H1 ES cell and HeLa 7SK ChIRP-seq. Chromatin samples were first treated with (+RNase) or without (-RNase) and recovery of human 7SK snrna and human 18S rrna were assessed. g, Number and classification (genic or distal) of ChIRP-seq peaks called from the three cell types examine are shown. In total 6,452, 5,512, and 12,006 7SK ChIRP-seq peaks were identified in H1, HeLa, and mouse ES cells. h, Scaled metagene analysis of 7SK ChIRP-seq from mouse ES (red), human ES (turquoise), and HeLa (green) cells. The upstream and downstream 1kb relative to the TSS and TTS, respectively, are shown. Gene bodies for each individual gene are scaled to the same length and average ChIRP-seq signal is plotted. i, Metagene analysis of 7SK ChIRP-seq in H1, HeLa, and mouse ES cells centered at active enhancer elements +/- 2kb.

4 Supplementary Figure 2 Factor ratio analysis relative to transcription initiation reveals promoter and enhancer differences. a, Mock example of how the factor ratio analysis is calculated for a given value of Factor X and TBP at a promoter and enhancer. b, Genome-wide correlation of ChIP-seq (13 factors), 7SK ChIRP-seq, and ATAC-seq datasets (Supplementary Table 7). 7SK clusters with factors marking active transcription. c, Extended factor ratio analysis of 41 ChIP-seq, 7SK ChIRP-seq, and ATAC-seq datasets (Supplementary Table 7). Log 2 fold change over TBP 54, TBP 55, or TAF1 55 ChIP-seq are shown from -2 (blue) to +2 (red). d, Heat maps of the 14,234 mrna promoters called using Start-seq data 31, sorted by the TSS-pair width, and oriented with the gene on the right. ChIP-seq signal for Pol II Ser5p, TBP, Nelf-a, ChIRP-seq signal of 7SK, and ATAC-seq is shown. e, Heat maps of the 361 individual SE peaks and 5356 TE regions containing Start-seq signal sorted by the total signal in each region (SE) or the TSS-pair width (TE). ChIP-seq and ChIRP-seq signal is as in (d). f, Principal component analysis using the datasets in (b) to classify promoters, SE, and TE.

5 Supplementary Figure 3 7SK depletion affects promoter and enhancer transcription differently. a, qrt-pcr analysis of 7SK snrna expression levels in mouse ES cells treated with ASOs. Levels are shown as a fraction of control knockdown cells and normalized to 18S rrna levels. Data are mean and s.d. of biological duplicate experiments. b,c, Volcano plot analysis of all promoters (b) and enhancers (c) with significant (FDR < 0.1) increased (red) or decreased (blue) GRO-seq after 7SK depletion. Quantification was performed to capture the area under the curve of change for each type of element: with promoters focusing on

6 the canonical Pol II paused region and enhancers focusing on the bidirectional region of transcription. d, Volcano plot analysis of all enhancers with significant (FDR < 0.1) increased (red) or decreased (blue) H3K36me3 ChIP-seq after 7SK depletion. e,f, GREAT analysis 57 of enhancer elements that gained significant GRO-seq (e) or H3K36me3 ChIP-seq (f) signal. The top 4 enriched terms are shown.

7 Supplementary Figure 4 Convergent transcription is gated by 7SK.

8 a, Schematic of GRO-seq emanating from two proximal TSS-pairs that would generate ConvT signal in the red highlighted area. b, Fold enrichment of convergent transcription (observed/expected) as determined by GROseq at promoters, SE, TE, Ctcf ChIP-seq peaks, and random genomic regions (Ten thousand, 2kb regions were randomly chosen from the genome 1,000 times. Each time, we calculate the Obs/Exp ratio of 100bp blocks that have higher than the mean CT). Specific enrichment values are noted above each bar. c, Comparison of ConvT levels at promoters, SE, and TE after 7SK depletion. Boxes represent 25 th and 75 th percentile and median values are plotted. K-S test *, p = 4.7x10-11, **, p < 2.2x d, Comparison of the GRO-seq read density (log 2, x-axis) and the change in convergent transcription after 7SK ASO (Fold Change 7SK ASO/Control ASO, log 2, y-axis). Values for all promoters (black), SE (blue), and TE (orange) are shown. e, Measurement of the distance between ATAC-seq peaks located within SE (green line) or TE (red line). f, Distribution of ConvT levels at the 2916 intergenic TE and 2319 intragenic TE elements. P value determined with K-S test of the difference between intergenic and intragenic TE. g, Comparison of γ-h2ax levels at promoters, SE, and TE after 7SK depletion. Boxes represent 25 th and 75 th percentile and median values are plotted. K-S test *, p = 1.7x10-8, **, p < 2.2x10-16.

9 Supplementary Figure 5 BAF and Hexim P-TEFb exist as two separate complexes, and 7SK does not modulate histone occupancy at promoters or enhancers. a, Co-IP study of the Hexim1 protein. Recovery of Hexim1, Arid1a, and Brg1 was determined via western blot in biological triplicate. b, Native RNA immunoprecipitation (RIP) qrt-pcr of Arid1a (BAF) or Hexim1 from mouse ES cells. Data are mean and s.d. of biological duplicate experiments. c,d, ChIP-seq analysis of pan-h3 occupancy changes due to 7SK loss at enhancers (c) and promoters (d). The Log 2 fold change (ASO/Scr) is shown, with significantly changed (FDR < 0.1). No elements were significantly changed in the H3 ChIP-seq experiment across two independent biological replicates.

11 Supplementary Figure 6 Depletion of 7SK causes defects in BAF-chromatin association. a,b, Glycerol gradient analysis of Control (black) or 7SK ASO (red) treated mes cells % glycerol gradients were prepared and 30 fractions were isolated from the top followed by western blot analysis. Canonical 7SK snrnp members (a) and Pol II or BAF subunits (b) are blotted. Shown are representative results of two independent experiments. c,d, Cumulative distribution function plot of Baf155 ChIP-seq enrichment values at SE (c) and TE (d). e, GREAT analysis 57 of enhancer elements that lost significant Baf155 ChIP-seq. The top 10 enriched terms are shown. f, Scatter plot (top) and individual contribution estimates (bottom) of three features (GRO-seq, Baf155 ChIP-seq, and ConvT) used in a multivariate linear model (Online Methods) that predicted the changes in γ-h2ax ChIP-seq signal after 7SK depletion.

12 Supplementary Figure 7 Binding of Pou5f1 and Sox2 is globally modulated by 7SK snrna. a, UCSC genome browser view of the sox2 locus with a proximal SE peak, marked with a pink bar. ChIP-seq

13 of Pou5f1 in control (black) or 7SK (red) ASO treated mouse ES cells, 7SK ChIRP-seq (orange) and ATAC-seq open chromatin (blue) are shown. b,c, Metagene analysis of Pou5f1 ChIP-seq at all enhancer elements (b) or promoters (c) in a +/-1kb window in mouse ES cells treated with control (black) or 7SK (red) ASOs. d,e, Cumulative distribution function plot of Pou5f1 ChIP-seq enrichment values at SE (d) and TE (e) elements. P- values were calculated with the K-S test. f, Transcription factor footprinting call scores from the PIQ algorithm ranked by footprint purity score 47. Scores for Ctcf (black), Pou5f1 (blue), and Sox2 (orange) are shown. g,h, Boxplot of Pou5f1, Sox2 (g) and Ctcf (h) footprint scores measured from ATAC-seq data with the PIQ algorithm. Samples treated with control or 7SK ASOs are shown as black and red, respectively.

14 Supplementary Figure 8 JQ1 alters 7SK-chromatin association independently of Pol II. a, qrt-pcr analysis of RNA enriched from mouse ES cell 7SK ChIRP-seq and the recovery of 7SK snrna and 18S rrna was assessed. b, qrt-pcr analysis of mouse ES cells treated with 500nM JQ1 for up to 6 hours. mrna expression levels were normalized to DMSO (mock, 0 min) treated cells. c, Metagene profile of 7SK ChIRP-seq in DMSO (black) or JQ1 (red) treated mouse ES cells across a scaled mrna locus. d,e,f, Metagene analysis of Pol II ChIP-seq in mouse ES cells at promoters (d), SE (e), and TE (f), colors are as in (c). g,h,i, Metagene analysis of 7SK ChIRP-seq in mouse ES cells at promoters (g), SE (h), and TE (i), colors are as in (c).

Comparison of open chromatin regions between dentate granule cells and other tissues and neural cell types.

Supplementary Figure 1 Comparison of open chromatin regions between dentate granule cells and other tissues and neural cell types. (a) Pearson correlation heatmap among open chromatin profiles of different