Virus- and interferon alpha-induced transcriptomes of cells from the microbat Myotis daubentonii

Supplementary Data

Martin Hölzer, Andreas Schoen, Julia Wulle, Marcel Müller, Christian Drosten, Manja Marz, and Friedemann Weber

Please cite:
Hölzer, M., Schoen, A., Wulle, J., Müller, M.A., Drosten, C., Marz, M., Weber, F., "Virus- and interferon alpha-induced transcriptomes of cells from the microbat Myotis daubentonii", iScience 19, 647-661 (2019)
https://doi.org/10.1016/j.isci.2019.08.016

Contents

Throughout the whole supplement, RVFV is used as an abbreviation for the Rift Valley fever virus Clone 13 mutant. Details can be found in the main manuscript.

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• Fig. S1: Myotis daubentonii kidney cell line MyDauNi/2c is fully IFN competent
• Table S1: Overview of all RNA-Seq data sets
• Table S2: STAR mapping rate
• Fig. S2: Principal component analysis
• Movies S1: Animated PCAs
• Fig. S3: Read classification (CLARK/Krona)
• Fig. S4: GO terms
• S4 A: Mock vs. Clone13
• S4 B: Mock vs. IFN
• S4 C: IFN vs. Clone13
• Fig. S5: DEG overview
• S5 A: Mock-6h vs. Mock-24h
• S5 B: Mock-6h vs. IFN-6h
• S5 C: Mock-6h vs. Clone13-6h
• S5 D: Mock-24h vs. IFN-24h
• S5 E: Mock-24h vs. Clone13-24h
• S5 F: IFN-6h vs. Clone13-6h
• S5 G: IFN-6h vs. IFN-24h
• S5 H: IFN-24h vs. Clone13-24h
• S5 I: Clone13-6h vs. Clone13-24h
• Fig. S6: RVFV genome coverage
• Fig. S7: RT-qPCR confirmation of virus-only response genes

Fig. S1: Myotis daubentonii kidney cell line MyDauNi/2c is fully IFN competent

(A) STAT1-dependent response to exogenous IFN. Human A549, M. daubentonii MyDauNi and R. aegyptiacus Ro6E-J cells where treated with IFN-α, the JAK1/2 inhibitor Ruxolitinib, or were left untreated. Immunoblot analysis was performed with antibodies against the indicated antigens. Representative data from three independent experiments are shown. (B) Multiplication of an IFN-sensitive virus. Cells were pretreated with IFN or Ruxolitinib, infected with the IFN inducing and IFN-sensitive RVFVΔNSs::Renilla, and virus replication was measured by Renilla luciferase assays after 48 h of incubation. The graph shows the virus reporter activities normalized to uninfected background values for each cell line. Mean values and standard deviations from three independent replicates are shown.

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Table S1: RNA-Seq data overview and preprocessing

Overview of all RNA-Seq data sets. All reads were quality checked with FASTQC and Prinseq. Ribosomal RNAs were removed by SortMeRNA. Shown are read statistics before and after trimming.

Nr	ID	Condition	Time	Replicate	# Reads (raw)	FastQC (raw)	# Reads (trimmed)	FastQC (trimmed)	% rRNA removed	5s	5.8s	18s	28s
1	N1_6h_mock_RNA	mock	6h	N1	72014608	FASTQC	71976311	TRIMMED	42.18	0.01	0.03	8.24	33.91
2	N2_6h_mock_RNA	mock	6h	N2	69174885	FASTQC	69136745	TRIMMED	42.15	0.01	0.02	8.43	33.68
3	N3_6h_mock_RNA	mock	6h	N3	66660208	FASTQC	66628120	TRIMMED	37.20	0.01	0.02	8.01	29.16
4	N1_24h_mock_RNA	mock	24h	N1	68994759	FASTQC	68967128	TRIMMED	16.29	0.01	0.02	3.14	13.11
5	N2_24h_mock_RNA	mock	24h	N2	67214554	FASTQC	67182327	TRIMMED	35.66	0.01	0.02	8.69	26.94
6	N3_24h_mock_RNA	mock	24h	N3	70693544	FASTQC	70653822	TRIMMED	65.44	0.01	0.03	17.34	48.07
7	N1_6h_IFN_RNA	IFN	6h	N1	70103253	FASTQC	70076333	TRIMMED	14.41	0.01	0.02	2.60	11.77
8	N2_6h_IFN_RNA	IFN	6h	N2	69501986	FASTQC	69467112	TRIMMED	30.24	0.01	0.02	6.16	24.04
9	N3_6h_IFN_RNA	IFN	6h	N3	70364742	FASTQC	70331915	TRIMMED	31.08	0.01	0.02	5.85	25.21
10	N1_24h_IFN_RNA	IFN	24h	N1	69072465	FASTQC	69043181	TRIMMED	17.84	0.01	0.02	3.24	14.56
11	N2_24h_IFN_RNA	IFN	24h	N2	66989439	FASTQC	66958823	TRIMMED	26.16	0.01	0.02	5.59	20.54
12	N3_24h_IFN_RNA	IFN	24h	N3	66867870	FASTQC	66831987	TRIMMED	64.45	0.01	0.03	15.21	49.20
13	N1_6h_RVFV_RNA	RVFV	6h	N1	68575488	FASTQC	68545199	TRIMMED	25.61	0.02	0.02	4.57	21.01
14	N2_6h_RVFV_RNA	RVFV	6h	N2	70292881	FASTQC	70259875	TRIMMED	28.91	0.01	0.02	5.96	22.91
15	N3_6h_RVFV_RNA	RVFV	6h	N3	71684876	FASTQC	71646117	TRIMMED	39.59	0.01	0.02	8.06	31.50
16	N1_24h_RVFV_RNA	RVFV	24h	N1	70118384	FASTQC	70092138	TRIMMED	15.17	0.02	0.03	2.33	12.79
17	N2_24h_RVFV_RNA	RVFV	24h	N2	68564186	FASTQC	68528972	TRIMMED	42.97	0.01	0.02	8.58	34.36
18	N3_24h_RVFV_RNA	RVFV	24h	N3	66246498	FASTQC	66212342	TRIMMED	39.92	0.01	0.02	8.80	31.09

Table S2: STAR mapping rate

We mapped the quality controlled and rRNA-depleted reads to the reference genome of the closely related microbat Myotis lucifugus using STAR.

(A) Uniquely mapped reads [%]. (B) Multiple mapped reads [%].

Nr	Sample	Input reads	Uniq mapped [%]	Uniq mapped [abs]	Number of splices	Mismatch rate per base [%]	Multiple mapped [%]	Multiple mapped [abs]	Unmapped (too short)
1	N1_6h_mock_RNA	41613966	70.69	29416823	4314735	2.05%	16.66	6931364	11.10%
2	N2_6h_mock_RNA	39998296	71.46	28583037	4197390	2.02%	16.66	6663829	10.35%
3	N3_6h_mock_RNA	41845008	71.64	29977924	4440966	2.03%	16.02	6702814	10.75%
4	N1_24h_mock_RNA	57732477	74.51	43014126	6490520	1.94%	15.44	8914894	8.57%
5	N2_24h_mock_RNA	43226582	73.06	31580887	4820715	1.97%	15.87	6858291	9.62%
6	N3_24h_mock_RNA	24415939	68.12	16632469	2370928	2.18%	16.69	4075183	13.69%
7	N1_6h_IFN_RNA	59981837	73.36	44002095	6673922	2.00%	16.55	9924770	8.54%
8	N2_6h_IFN_RNA	48461374	72.9	35329169	5292556	2.02%	16.44	7968739	9.17%
9	N3_6h_IFN_RNA	48470406	72.36	35074137	5267434	2.03%	16.3	7899697	9.82%
10	N1_24h_IFN_RNA	56724654	74.43	42221426	6284716	1.99%	14.93	8470254	9.11%
11	N2_24h_IFN_RNA	49439582	74.48	36824901	5661325	1.96%	15.76	7792275	8.28%
12	N3_24h_IFN_RNA	23760050	69.25	16453496	2353279	2.18%	16.33	3879685	12.88%
13	N1_6h_RVFV_RNA	50991652	73.17	37311443	5355209	1.98%	16.21	8266558	9.08%
14	N2_6h_RVFV_RNA	49950316	73.08	36503929	5405391	1.99%	16.32	8151527	9.12%
15	N3_6h_RVFV_RNA	43282410	72.39	31330065	4552946	2.03%	16.0	6925579	10.02%
16	N1_24h_RVFV_RNA	59461224	73.8	43884200	5978846	2.02%	14.09	8375445	10.63%
17	N2_24h_RVFV_RNA	39081917	73.23	28619885	3873904	2.07%	14.89	5819071	10.37%
18	N3_24h_RVFV_RNA	39781791	73.75	29337729	4239232	2.06%	15.16	6030454	9.60%

Fig. S2: Principal component analysis

A: PCA transformation based on the top 500 variant genes over all samples. One of the 24h RVFV mutant samples seems to not cluster that well with the other replicates.	B: PCA transformation based on the top 500 variant genes between mock 24h and RVFV mutant 24h samples. The difference between the "outlier" virus-infected sample and the other two replicates accounts only for 3.3% (PC2) of the whole variation in the gene expression data.

Movies S1: Animated PCAs

(A) animated 2d-PCA and (B) animated 3d-PCA movies generated with PCAGO.

A:
Show animated 2d-PCA movie in browser: mp4 (744K)
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B:
Show animated 3d-PCA movie in browser: mp4 (2.3M) | mov (44Mb)
Download animated 3d-PCA movie: mp4 (2.3M)
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Fig. S3: Read classification with CLARK and visualization with Krona

CLARK classifcation was run against NCBI RefSeq genomes of bacteria, viruses, fungi, human and a custom database comprising the M. lucifugus genome. Click on the "link" button to copy the URL and open the circular plot in a larger view.

Mock 6h

Mock 24h

IFN 6h

IFN 24h

Clone13 6h

Clone13 24h

Fig. S4: Enriched GO terms based on pairwise comparisons

The calculation of significantly enriched GO terms is based on the Piano calculations that can be found in the corresponding DEG calculations (see S5 A-I in overview).

Fig. S4 A: Mock vs. Clone13

GO terms of the virus-regulated genes. Significantly enriched GO categories for the mock vs. Clone 13 comparisons 6 h (upper) and 24 h (lower) post infection. Based on the Piano output (see supplementary data S5 C and S5 E), significantly enriched GO categories (p<0.05) composed of at least five differentially expressed genes were selected. The bars indicate how many genes are significantly up (red) or down (blue) regulated within a specific GO category. The GO categories are sorted by "biological process" (green), "molecular function" (blue), and "cellular component" (orange) in ascending order regarding the number of genes. Download PDF

Fig. S4 B: Mock vs. IFN

GO terms of the IFN-regulated genes. Significantly enriched GO categories for the mock vs. IFN comparisons 6h (upper) and 24h (lower) post infection. Based on the Piano output (see supplementary data S5 B and S5 D), significantly enriched GO categories (p<0.05) composed of at least five differentially expressed genes were selected. The bars indicate how many genes are significantly up (red) or down (blue) regulated within a specific GO category. The GO categories are sorted by "biological process" (green), "molecular function" (blue), and "cellular component" (orange) in ascending order regarding the number of genes. Download PDF

Fig. S4 C: IFN vs. Clone13

GO terms of the IFN- and Clone 13-regulated genes. Significantly enriched GO categories for the IFN vs. Clone 13 comparisons 6h (upper) and 24h (lower) post infection. Based on the Piano output (see supplementary data S5 F and S5 H), significantly enriched GO categories (p<0.05) composed of at least five differentially expressed genes were selected. The bars indicate how many genes are significantly up (red) or down (blue) regulated within a specific GO category. The GO categories are sorted by "biological process" (green), "molecular function" (blue), and "cellular component" (orange) in ascending order regarding the number of genes. Download PDF

Fig. S5: DEG overview

Sample2Sample distance

Heatmaps

Top genes with highest expression	Genes up-regulated only in RVFV 24h

All pairwise comparisons:

• S5 A: Mock-6h vs. Mock-24h
• S5 B: Mock-6h vs. IFN-6h
• S5 C: Mock-6h vs. Clone13-6h
• S5 D: Mock-24h vs. IFN-24h
• S5 E: Mock-24h vs. Clone13-24h
• S5 F: IFN-6h vs. Clone13-6h
• S5 G: IFN-6h vs. IFN-24h
• S5 H: IFN-24h vs. Clone13-24h
• S5 I: Clone13-6h vs. Clone13-24h

Fig. S6: Coverage of the S, M, and L segment of the RVFV reference genome

Reference sequences for Clone 13 of the S, M, and L segment of RVFV were downloaded from the NCBI under RefSeq accessions NC014395.1 (S segment), NC014396.1 (M segment), NC014397.1 (L segment). The plots show the aggregate average coverage for the triplicates 6h and 24h post infection with the RVFV Clone13 mutant.

Please note the Clone 13-specific deletion in the IFN antagonist NSs on the S segment that determines its high IFN induction capability.

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Fig. S7: RT-qPCR confirmation of virus-only response genes - control experiments

Human A549 and M. daubentonii MyDauNi cells, treated or not with Ruxolitinib, were incubated with 1000 U/ml IFN-α or infected with Clone 13 (MOI 5) for 6 or 24 h, respectively. (A) Immunoblot analysis was performed with antibodies against the indicated antigens. Representative data from three independent experiments are shown. (B) RT-qPCR for MxA/Mx1 transcripts and the viral L segment, respectively. The graphs show the fold induction over mock, for the respective time point, with mean values and standard deviations from three independent replicates.

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