SUPT4H1 Depletion Leads to a Global Reduction in RNA Adam Naguib, Thomas Sandmann, Fei Yi, Ryan J. Watts, Joseph W. Lewcock, William E. Dowdle  Cell Reports  Volume 26, Issue 1, Pages 45-53.e4 (January 2019) DOI: 10.1016/j.celrep.2018.12.004 Copyright © 2018 Denali Therapeutics Inc Terms and Conditions

Cell Reports 2019 26, 45-53.e4DOI: (10.1016/j.celrep.2018.12.004) Copyright © 2018 Denali Therapeutics Inc Terms and Conditions

Figure 1 Use of Spike-In Standards to Measure Global Changes in RNA Abundance by RNA Sequencing (A) Replicate scheme for RNA sequencing. (B) Experimental workflow illustrating siRNA knockdown, harvesting, and spike-in of ERCC RNA standards for RNA sequencing. (C) qRT-PCR confirmation of SUPT4H1 knockdown in HEK293 cell line samples submitted for RNA sequencing. Two independent transfections for each condition were assessed by qRT-PCR in triplicate. Error bars, SD. RQ, relative quantification. (D) Comparison of external standard abundances between replicates. Normalized abundance of ERCC spike-in transcripts (counts per million [CPM]) values were plotted to assess consistency across technical replicates. Low abundance transcripts detected in only a single replicate are depicted as tick marks on axes. Cell Reports 2019 26, 45-53.e4DOI: (10.1016/j.celrep.2018.12.004) Copyright © 2018 Denali Therapeutics Inc Terms and Conditions

Figure 2 SUPT4H1 Knockdown Leads to Global Reduction in Non-rRNA (A) Data from EGFP control and DARP cell lines were combined and binned by treatment (n = 6 scramble and n = 6 SUPT4H1 siRNA conditions), and observed expression changes between SUPT4H1 and scramble RNAi conditions are depicted (cellular RNA). Observed fold change for synthetic spike-in standards is also shown. The x axis is the expected fold change (log2) for each group of synthetic standards. Fold change in expression was normalized using synthetic spike-in standards. Boxplots display the median (second quartile) and the interquartile (first and third) range. Whiskers represent the lowest and highest data within 1.5× of the interquartile range from the first and third quartiles. (B) MA plot showing transcript abundances after SUPT4H1 knockdown. Spike-in standards are depicted by colored dots, with corresponding lines representing the median log2 fold change between SUPT4H1 and scramble siRNA treated conditions for each subgroup of ERCC controls. Density of endogenous transcripts is shown in blue (normalized to spike-in standards). (C) Fold change in cellular RNA (SUPT4H1 knockdown versus control) normalized using synthetic spike-ins, stratified by cell line. Observed abundance of synthetic spike-in standards is also shown. The x axis shows expected fold change (log2) of the standards. Box plots as in (A). (D) Normalized CPM for EGFP transcript abundances. CPM are shown for scramble and SUPT4H1 siRNA treated conditions. Bars, SD. (E) Left: expression construct architecture for intron-containing 71 HNR expression plasmid. Immunoblot detection of 71 HNR-derived DARP. Equivalent masses of 71 HNR plasmid encoding or absent 0.16 kb of the endogenous human C9orf72 intronic sequence were independently transfected into HEK293 cells and 24 hr later harvested for immunoblot comparison of DARP expression. Right: fluorescent light microscopy determination of subcellular distribution of DARPs expressed from constructs with or without C9orf72 intronic sequence. Scale bars, 200 μm. DARP expression is detectable, although in a minority of cells when expressed from intron-containing constructs. DARP aggregates (asterisks) are observed in cells transfected with either construct. (F) Confocal fluorescence microscopy of DARP aggregates in clonal HEK293 cells derived from the integration of 71 HNR constructs encoding human C9orf72 intronic DNA. Scale bar, 20 μm. (G) Fold change in RNA expression normalized using synthetic spike-ins for clonal HEK293 cells derived from the integration of the C9orf72 intron-containing 71 HNR constructs after SUPT4H1 RNAi-mediated knockdown. Expression fold change (SUPT4H1 knockdown versus control) is depicted. Box plots as in (A). (H) Normalized CPM for EGFP transcript abundances in HEK293 cells derived from the integration of 71 HNR constructs encoding human C9orf72 intronic DNA. CPM are shown for scramble and SUPT4H1 siRNA treated conditions. Bars, SD. (I) Exon-intron split analysis (EISA) comparing mean log2 fold changes of intronic and exonic alignments (spike-in normalized). Cell Reports 2019 26, 45-53.e4DOI: (10.1016/j.celrep.2018.12.004) Copyright © 2018 Denali Therapeutics Inc Terms and Conditions

Figure 3 Cellular RNA Is Diminished after SUPT4H1 Knockdown in Multiple Cell Types (A) qRT-PCR assessment of SUPT4H1 knockdown in HeLa cells. Three independent wells for each RNAi treatment were each measured in triplicate. Error bars, SD. RQ, relative quantification. (B) SYTO RNASelect dye quantitation of cellular RNA content after SUPT4H1 knockdown in HeLa cells. Nuclear and cytoplasmic compartments were assayed and fluorescence intensity measured. Mean fluorescent intensity per cell, stratified by compartment, is depicted. Dye distributions are represented in the micrograph. Images shown are HeLa cells treated with scramble siRNA. p values: t test, 2-tailed, equal variance. Scale bar, 50 μm. Error bars, SD of the mean from 3 independent wells. (C) qRT-PCR confirmation of SUPT4H1 knockdown in HeLa and A549 cells (3 independent wells for each RNAi treatment were each measured in triplicate) and SYTO RNASelect dye intensities, per cell, in nuclear (center) or cytoplasmic (bottom) compartments. Fluorescent intensities are normalized to the scramble treated cells to allow comparison across cell lines. p values: t test, 2-tailed, equal variance. Error bars, SD of the mean from 3 independent wells. RQ, relative quantification. (D) Density plot of flow cytometry analyzed live-HEK293 cells treated with SYTO RNASelect dye. Scramble and SUPT4H1 siRNA treatments are shown (3 days, 100 nm). (E) qRT-PCR confirmation of SUPT4H1 knockdown in HeLa cells using alternative siRNA oligonucleotides (left); error bars, SD of the mean. Three independent wells for each RNAi treatment were measured in triplicate. SYTO RNASelect dye quantitation of per-cell RNA content in the nuclear (upper right) and cytoplasmic (lower right) compartments post-knockdown and RNase treatment. Error bars, SD of the mean. Replicates from 8 wells for each condition were measured by high-content imaging, except for RNase conditions, in which 4 wells were assayed per condition. p values: t test, 2-tailed, equal variance. RQ, relative quantification. Cell Reports 2019 26, 45-53.e4DOI: (10.1016/j.celrep.2018.12.004) Copyright © 2018 Denali Therapeutics Inc Terms and Conditions

Figure 4 SUPT4H1 Knockdown in Primary, Non-immortal Human Cells Causes a Global Reduction in mRNA (A) Percentage coefficient of variance (%CV) values for fibroblast cell counts. %CV values were determined from 2 replicate counts of each sample. For each experimental condition, 3 independent %CV values for each independent treatment are displayed. (B) qRT-PCR confirmation of SUPT4H1 knockdown in human fibroblasts. Error bars, SD of the mean. RQ, relative quantification. Three independent transfections for each RNAi treatment were each measured in duplicate or triplicate. (C) Transcript abundance fold changes in fibroblasts after SUPT4H1 knockdown. Expression was normalized using synthetic spike-in standards. Boxplots display the median (second quartile) and interquartile (first and third) range. Whiskers represent the lowest and highest data within 1.5× of the interquartile range from the first and third quartiles. SUPT4H1 fold change (treatment versus control) is depicted. Expected standard fold changes are shown (x axis). SUPT4H1 siRNA treatments, 5 and 10 nM, are shown in independent plots. (D) Compiled data from both siRNA fibroblast treatments (n = 3 scramble, n = 6 SUPT4H1 siRNA). Box plots as in (C). Cell Reports 2019 26, 45-53.e4DOI: (10.1016/j.celrep.2018.12.004) Copyright © 2018 Denali Therapeutics Inc Terms and Conditions