1

Cross-species comparative analysis of Dicer proteins during Sindbis

virus infection

Erika Girardi1, 3, Mathieu Lefèvre1, Béatrice Chane-Woon-Ming1, Simona Paro2, Bill

Claydon2, Jean-Luc Imler2, Carine Meignin2, *, Sébastien Pfeffer1, *

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY METHODS

Constructs used in the study

A PCR fragment corresponding to the whole Dicer-2 cDNA sequence was amplified

from Dicer-2 full-length pOT2-cDNA clone (derived from SD11113 clone) using the

primers indicated in OLIGO LIST. Dicer-2 was cloned into the pENTR/D-TOPO

Gateway entry vector using the pENTR directional TOPO cloning kit (Invitrogen) for

N-terminal fusion. pFRT/TO/FLAG/HA-DEST DICER vector (Addgene) was used as

starting template to generate by recombination pDONOR-ZEO-hDicer.

The Dicer-2 and hDicer cDNA were then transferred to Drosophila transgenic

destination vector pURW (a kind gift from Jean-René Huynh’s laboratory,

DGRC#1282) by LR recombination using the Gateway LR clonase II enzyme mix

(Invitrogen) and used for transgenic flies generation.

To obtain pFlag-HA-Dicer-2-Puro, we recombined the pDONOR-Dicer-2 with the

destination vector pDEST Flag-HA vector using the Gateway cloning system. To

obtain pIRES-V5-R2D2/myc-Ago2-Neo, R2D2 and Ago2 genes were amplified by

  2

PCR (See OLIGO LIST) and cloned into pIRES vector (cat# 631605 Clontech).

pFlag-HA-R2d2 and pFLAg-HA-Ago2 have been generated as for pFlag-HA-Dicer2

and only used for transient transfection experiments.

Fly maintenance and infection conditions

Flies were fed on standard cornmeal–agar medium at 25 ̊C. All fly lines were tested

for Wolbachia infection and cured whenever necessary. Infections were performed

with 3- to 5-days old adult flies by intrathoracic injection (Nanoject II apparatus;

Drummond Scientific) with viral particles. Sindbis viral stock was prepared in 10 mM

Tris-HCl, pH 7.5 [SINV 5x108 PFU/ml]. Injection of the same volume (4.6 nL) of 10

mM Tris-HCl, pH 7.5, was used as a control. Infected flies were then incubated at

25°C, and monitored daily for survival, or frozen for RNA isolation and virus titration

at 5 days post infection (dpi).

Viral stocks, cell culture and virus infection

TE3’2J infectious clone containing SINV genomic sequence was linearized with XhoI

and used as a substrate for in vitro transcription using mMESSAGE mMACHINE

capped RNA transcription kit (Ambion) following the manufacturer’s instructions.

Sindbis viral stock was prepared in BHK21 hamster kidney cells and titrated by

plaque assay. HEK293 cells were maintained in Dulbecco's modified Eagle medium

(DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Clontech) in a

humidified atmosphere of 5% CO2 at 37°C. Cells were infected with SINV at MOI

0.01 (except when indicated differently) and samples were collected at different time

points as indicated in the figure legends.

  3

RNA Extraction and Real-Time PCR analysis

For the human samples, total RNA was isolated using TRI-Reagent (Ambion,

AM9738) and 2 μg of RNA were DNaseI treated and reverse transcribed with

SuperScript III (Invitrogen, Ref: 18080-080) according to manufacturer’s instructions.

Quantitative real-time PCR was performed using Maxima SYBR Green (Fermentas,

#K0253), on a CFX96 Touch™ Real-Time PCR Detection System (Biorad). Gene

expression was normalized to beta actin.

Total RNA from infected flies was isolated using TRI Reagent RT bromoanisole

solution (MRC) and reverse transcribed using iScript cDNA Synthesis Kit (Bio-Rad)

on the T3000 Thermocycler (Biometra). Quantitative real-time PCR was performed

using the iQ Custom SYBR Green Supermix Kit (Bio-Rad) on the CFX384 Real-

Time System (Bio-Rad). Gene expression was normalized to RP49.

All the primers used are listed in the OLIGO LIST.

Statistical analysis

An unpaired two-tailed Student t-test was used for statistical analysis of data with

GraphPad Prism (GraphPad Software). The p-values < 0.05 were considered

statistically significant. Survival curves were plotted and analyzed by log-rank test

using GraphPad Prism (GraphPad Software).

RNAi-mediated protein depletion

SiRNA transfection was performed using the reverse transfection method. Briefly,

siRNAs (100 nM, final concentration) were incubated with lipofectamine 2000

(Invitrogen) as described by the manufacturer. Transfection mix was incubated to the

cells in suspension. Two days later, transfected cells were infected with SINV MOI

  4

0.01. Sixteen hours post-infection, cells were lysed for RNA analysis. SiRNAs used

were ON-TARGET plus Smart Pool siRNAs (Dharmacon) targeting human RNaseL

(L-005032-01), PKR (L-003527-00), RIG-I (L-012511-00), MDA5 (L-013041-00),

hDicer (L-003483-00) and a siRNA control (D-001810-10).

Western-blot and immunostaining analysis

Proteins were extracted from 10 females of each genotype and homogenized in 200 µl

of lysis buffer (30 mM Hepes, 150 mM NaCl, 2 mM Mg(OAc)2, 5 mM DTT, 0.1%

NP40 and protease inhibitor ‘cocktail’ (cOmplete Mini; Roche Diagnostics)) with the

Precellys®24 Dual, according to the manufacturer’s instructions. Proteins were

quantified by the Bradford method and 50 µg of total protein extract was loaded on 4-

20% Mini-PROTEAN® TGX™ Precast Gels (Biorad). After transfer onto

nitrocellulose membrane, equal loading was verified by Ponceau staining. Membranes

were blocked in 5% milk. The membrane was probed with the following antibodies:

anti-Dicer2 (1:1000, anti-PAZ domain, as indicated in Fig S1A), anti-hDicer (1:1000,

Bethyl,#A301-936A ), anti-R2D2 (1:2000, 1), anti-V5 (1:5000, Abcam, ab9116), anti-

drosophila Argonaute 2 (kind gift of Prof. M. Siomi), anti-HA (1:10000,   Sigma,

H9658) and anti-Actin clone 4 (1:5000, Millipore #MAB1501R). Detection was

performed using Chemiluminescent Substrate (Amersham). Immunofluorescence

experiments were carried out on cells infected with SINV-GFP (MOI 1) to guarantee

that each cell was infected with 1 viral particle. Given the observed cell death at 16hpi

using MOI 1, we chose the 12hpi time point for this experiment.

Mouse anti-FLAG (1:500, Sigma F-3165), rabbit anti-V5 (1:500, Abcam ab9116),

mouse Anti-dsRNA J2 (1:200, ESC- 10010200), Alexa Fluor® 488 anti-mouse

(1:500, Molecular Probes® A-21202), Alexa Fluor® 568 anti-rabbit (1:500,

  5

Molecular Probes® A10042) and DAPI to visualize cell nuclei. Images were taken

using Spinning disc Zeiss. ImageJ was used to analyze the cells.

IFNβ promoter-luciferase assay and IFNβ mRNA induction assay

The promoter region of the human IFNβ gene was amplified from genomic DNA and

cloned upstream of the firefly luciferase gene in the pGL4-basic vector. The resulting

plasmid, designated pGL4-IFNβ-luc, was transfected in the HEK293 stable cell lines

in 96 well plates using lipofectamine 2000 (Invitrogen) according to the manufacturer

instructions. Two days post-transfection, cells were incubated with free opti-MEM

(untreated) or dsRNA poly I:C (Invivogen, tlrl-picw) at the indicated concentrations.

Twenty-four hours after treatment, cells were lysed and IFNβ promoter activity was

determined using a luciferase assay (Promega, Dual Luciferase Reporter Assay,

E1960) with a luminometer (Promega, GloMax® Multi Detection System). Absolute

firefly luciferase activity was normalized for transfection efficiency using the

untreated cells.

For the detection of the IFNβ and IL8 mRNA induction, cells were transfected with

either poly I:C (20µg/mL) using with lipofectamine 2000 (Invitrogen) as described by

the manufacturer or treated with Flagellin (100ng/mL; Flagellin from S. typhimurium,

InvivoGen) or IL-1β (10ng/mL; Recombinant human interleukin-1beta, InvivoGen).

Cells were lysed 6 hours and total RNA was extracted and retrotranscribed as

mentioned above.

  6

SUPPLEMENTARY FIGURE LEGENDS

Figure S1: Generation of RFP::hDicer and RFP::Dicer-2 transgenic flies. A)

Schematic representation of Dicer-2 and hDicer domain architecture. The Helicase

domain contains the DExD/H, DExD/DExH box helicase domain and HELiCc,

helicase conserved C-terminal domain. dsRBD, double-strand RNA binding domain;

PAZ, PAZ domain; RIIIa and RIIIb, ribonuclease III domains. Platform-PAZ-

connector helix was defined by 2. L811fsX represents the null allele of dicer-2 used in

this study. Peptide 849-866 was used to produce the Dicer-2 antibody. B) Genetic of

Dicer-2 and hDicer transgenic lines. Dicer-2 gene is localized in the 2nd-chromosome

in 2R, 54C10. The Deficiency Df(2R)BSC45 covers the cytologic map from 2R,

54C8 to 2R, 54E7. The Dicer-2 genomic rescue contains the full dicer-2 gene. The

lower panels schematically represent the cytologic position of each transgene used.

Figure S2: Effect of RFP::hDicer and RFP::Dicer-2 expression on D.

melanogaster endogenous siRNA pathway. Eye color of the following genotypes:

(1) CantonS (wt), (2), wIR; dicer-2L811fsX/Df, (3) wIR; dicer-2L811fsX/Df-Rescue,

(4) wIR; dicer-2L811fsX/Df-RFP::Dicer-2, (5) wIR; dicer-2L811fsX/Df-

RFP::hDicer.

Figure S3: Cellular miRNA profiling in SINV-infected transgenic flies. The

heatmap shows hierarchical clustering of the different samples and of the 100 most

abundant miRNAs in each sample on the basis of their expression profile. High

relative miRNA expression (log2-transformed) is indicated by blue shades, low

expression by green shades.

  7

Figure S4: Effect of RFP::hDicer expression on Drosophila melanogaster innate

immune response. Relative mRNA level of vago (Dicer-2-dependent), diptericin

(IMD pathway), drosomycin (Toll pathway), TotM (Jak/STAT pathway) and vir-1

(viral infection related) compared to the housekeeping gene rp49 in flies upon SINV

infection. The expression level of each gene is normalized to the genotype wIR; dcr2-

/Df, Rescue. Data are representative of at least three independent experiments

(average and SEM) with 6 flies of each genotype. T-test was performed between the

control genotype wIR; dcr2-/Df, Rescue and Dicer-2 null (wIR; dcr2-/Df) or

RFP::Dicer-2 (5A) and RFP::hDicer (1A and 1D). ns (non-significative), *p<0.05,

**p<0.01, ***p<0.001. All experiments were performed at 25°C and flies were

collected 5 days post-infection by SINV at 2500 plaque forming units (pfu).

Figure S5: R2D2, Dicer2 and dAgo2 can be individually expressed in HEK293

cells. Left panel Schematic representation of the plasmids used for transient

transfection in HEK293 cells. The plasmid pFlag-HA-Dicer-Puro, pFlag-HA-Ago2-

Puro and pFlag-HA-R2D2-Puro drives the expression of Dicer-2, Ago2 and R2D2,

respectively. Right panel Western blotting to measure Flag-HA-Dicer2, -R2D2, and -

dAgo2 protein expression levels in HEK293 cell lines. Anti-HA antibody was used.

Ponceau staining was used as loading control. *, ** and *** indicate the expected size

for Dicer2, Ago2 and R2d2 respectively. – indicates the mock transfected control.

Figure S6: Cellular miRNA profiling in SINV infected human cell lines. The

heatmap shows hierarchical clustering of the different samples and of the 100 most

abundant miRNAs in each sample on the basis of their expression profile. High

  8

relative miRNA expression (log2-transformed) is indicated by blue shades, low

expression by green shades.

Figure S7: Deep sequencing of viral sRNAs in SINV infected HEK293 stable cell

lines at 16 hours post infection. Size distribution of SINV-derived sRNA

populations.

Figure S8: Comparison of 21-nt long viral sRNAs in SINV infected HEK293

stable cell lines at both 6 and 16 hours post infection. Coverage of the 21-nt viral

reads was calculated and plotted as the sum of normalized reads (RPM, Reads Per

Million mapped reads) in each single-nucleotide sliding window along the SINV

genome. A schematic diagram represents the organization of SINV genome. The red

bar corresponds to the first 1500 nt (nsp1) of SINV genome, as shown in Figure 3 and

S7. Positive (+) and negative (-) strand-derived reads are shown in blue and orange,

respectively.

Figure S9: Accumulation of dsRNA during Sindbis infection. HEK293e, Dicer-2-

HEK293 and DAR-HEK293 stable cell lines were immunostained with anti-dsRNA

J2 antibody (magenta). Immunostaining was performed at 0, 6, 12 hours post SINV or

(MOI 1). DAPI staining is shown in blue.

Figure S10: Activation of the IFNβ promoter in HEK293e, Dicer-2- and DAR-

HEK293 cells. Cells were transfected with pGL4-IFN-β-firefly luciferase constructs

and were treated with polyI:C (5, 10, 20 µg/ml). Relative luciferase was measured

  9

compared to unchallenged condition. All observed differences were significant (t-test

p<0.05).

  10

SUPPLEMENTARY REFERENCES

1. Nishida, K. M. et al. Roles of R2D2, a Cytoplasmic D2 Body Component, in the Endogenous siRNA Pathway in Drosophila. Molecular Cell 49, 680–691 (2013).

2. MacRae, I. J. Structural Basis for Double-Stranded RNA Processing by Dicer. Science 311, 195–198 (2006).

  11

SUPPLEMENTARY TABLE

List of oligonucleotides used in this study

qRT-PCR b-Actin fw TTTGAGACCTTCAACACCCCA b-Actin rv TTTCGTGGATGCCACAGGA diptericin fw GCTGCGCAATCGCTTCTACT diptericin rv TGGTGGAGTGGGCTTCATG drosomycin fw CGTGAGAACCTTTTCCAATATGATG drosomycin rv TCCCAGGACCACCAGCAT EIF2AK2(PKR) fw TGGAAAGCGAACAAGGAGTAAG EIF2AK2(PKR) rv CCAAAGCGTAGAGGTCCACTT GAPDH fw CTTTGGTATCGTGGAAGGACT GAPDH rv CCAGTGAGCTTCCCGTTCAG hRIG-I (DDX58) fw TCCTTTATGAGTATGTGGGCA hRIG-I (DDX58) rv CCAGCATTACTAGTCAGAAGG IFNb fw GTCTCCTCCAAATTGCTCTC IFNb rv ACAGGAGCTTCTGACACTGA IL8 fw ACTGAGAGTGATTGAGAGTGGAC IL8 rv AACCCTCTGCACCCAGTTTTC MDA-5 (IFIH1) fw GCCCGCTACATGAACCCTG MDA-5 (IFIH1) rv CAGCAATCCGGTTTCTGTCTT RNaseL fw GTAAACGCCTGTGACAATATGGG RNaseL rv AGATGCGTAATAGCCTCCACAT RP49 fw GACGCTTCAAGGGACAGTATCT RP49 rv AAACGCGGTTCTGCATGAG SINV fw CCACTACGCAAGCAGAGACG SINV rv AGTGCCCAGGGCCTGTGTCCG SINV fw (fly) CAAATGTGCCACAGATACCG SINV rv (fly) ATACCCTGCCCTTTCAACAA TotM fw GCTGGGAAAGGTAAATGCTG TotM rv AGGCGCTGTTTTTCTGTGAC Vago fw TGCAACTCTGGGAGGATAGC- Vago rv AATTGCCCTGCGTCAGTTT vir-1 fw GATCCCAATTTTCCCATCAA vir-1 rv GATTACAGCTGGGTGCACAA CLONING myc-dAgo2 fw CGGTCTAGAATGGAACAAAAACTTATTTCTGAAGAAGATCTCGGAAAAAAAGATAAGAAC myc-dAgo2 rv CCGGTCGACTTATCACTATCAGACAAAGTACATGGG V5-R2D2 fw CGCGCTAGCATGGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGGATAACAAGTCAGCCGTA V5-R2D2 rv CGCCTCGAGTTATCACTATTAAATCAACATGGTGCG Dicer-2 cDNA fw GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGATGTGGAAATCAAGCCTCGCG Dicer-2 cDNA rv GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATCACTATTAGGCGTCGCATTTGCTTAGCTGC

Chromosome 2R: 13,462,484-13,469,031 reverse strand.

CG6493 (dcr-2) CG6484CG4921 (Rab-4)

1kb

#BL7441 Df(2R)BSC45 [w+]

2R, 54C10

2R, 54C8 2R, 54E7

Dicer-2 genomic rescue - FlyFos017074 (32,706 kb) 2R, 54D1

2L 2R

Rescue

25C7 54C10dcr-2Genomic rescue

2L 2R

21B

54C10dcr-2Transgenic lines

6

5

1

43A

57B

Genomic rescue inserted at attP40 (25C7)

insertion in (+) orientation

insertion in (-) orientation

RFP::Dicer-2

RFP::hDicer

ARIIIa RIIIb

dsRBDdsRBDDExD/H HELICcHelicase Platform PAZ

Connectorhelix

1722 aaFly Dicer-2

1922 aaHuman Dicer RIIIa RIIIbdsRBD

dsRBDDExD/H HELICcHelicase Platform PAZ

Connectorhelix

L811fsx

B

peptide 849-866

Girardi et al. Fig S1

1

2

3

4

5

Girardi et al. Fig S2

SINV−Df

SINV−RFP::Dicer−2

SINV−RFP::hDicer

dme−miR−310−3pdme−miR−994−5pdme−miR−999−3pdme−miR−14−5pdme−miR−987−5pdme−miR−275−5pdme−miR−133−3pdme−miR−307a−3pdme−miR−1010−3pdme−miR−281−1−5pdme−miR−31b−5pdme−miR−79−3pdme−miR−1000−5pdme−miR−33−3pdme−miR−1003−3pdme−miR−284−5pdme−miR−193−5pdme−miR−13b−2−5pdme−miR−11−5pdme−miR−1006−3pdme−miR−137−3pdme−miR−285−3pdme−miR−13a−3pdme−miR−2c−3pdme−miR−1012−5pdme−miR−282−5pdme−miR−993−3pdme−miR−932−5pdme−miR−2b−2−5pdme−miR−996−5pdme−miR−317−5pdme−miR−305−3pdme−miR−958−5pdme−miR−87−3pdme−miR−965−3pdme−miR−988−3pdme−miR−1012−3pdme−miR−124−3pdme−miR−9a−3pdme−miR−276a−5pdme−miR−276b−5pdme−miR−34−5pdme−miR−14−3pdme−miR−277−3pdme−miR−184−3pdme−miR−276a−3pdme−miR−1−3pdme−miR−8−3pdme−bantam−3pdme−miR−317−3pdme−miR−305−5pdme−let−7−5pdme−miR−210−3pdme−miR−989−3pdme−miR−2b−3pdme−miR−956−3pdme−miR−8−5pdme−miR−314−3pdme−miR−13b−3pdme−miR−252−5pdme−miR−283−5pdme−miR−10−3pdme−miR−10−5pdme−miR−9a−5pdme−miR−31a−5pdme−miR−281−2−5pdme−miR−281−3pdme−miR−275−3pdme−miR−375−3pdme−miR−263a−5pdme−miR−306−5pdme−miR−970−3pdme−miR−958−3pdme−miR−279−3pdme−miR−311−3pdme−miR−957−3pdme−miR−276b−3pdme−bantam−5pdme−miR−312−3pdme−miR−190−5pdme−miR−92a−3pdme−miR−125−5pdme−miR−316−5pdme−miR−34−3pdme−miR−9c−5pdme−miR−11−3pdme−miR−996−3pdme−miR−278−3pdme−miR−981−3pdme−miR−7−5pdme−miR−33−5pdme−miR−318−3pdme−miR−304−5pdme−miR−2a−3pdme−miR−986−5pdme−miR−274−5pdme−miR−9b−5pdme−miR−12−5pdme−miR−995−3pdme−miR−92b−3p

8 14

Girardi et al. Fig S3

TotMdiptericin drosomycinvago

0

2

4

6

ns

0

2

4

6 **

0

2

4

6

ns

0.0

0.5

1.0

1.5ns

ns

vir-1

0

1

2

3

4

**ns

Rel

ativ

e m

RN

A Ex

pres

sion

* **ns

*

wIR; dcr2-/Df, Rescue wIR; dcr2-/Df wIR; dcr2-/Df, RFP::Dicer-2 wIR; dcr2-/Df, RFP::hDicer

Girardi et al. Fig S4

Anti-HA

Ponceau

Flag-H

A-Ago

2-Puro

Flag-H

A-Dice

r2-Puro

Flag-H

A-R2d

2-Puro

-

250 kDa130 kDa100 kDa

35 kDa

70 kDa55 kDa

***

***

R2d2

Dicer-2Flag-HA

Flag-HA

Flag-HA

CMV

CMV

CMV

pFlag-HA-Dicer2-Puro

pFlag-HA-Ago2-Puro

pFlag-HA-R2d2-Puro

Ago2

Puro

Puro

Puro

Girardi et al. Fig S5

Dicer−2−HEK293.16hpi

DAR−HEK293.16hpi

HEK293

e.16hpi

Dicer−2−HEK293.6hpi

DAR−HEK293.6hpi

HEK293

e.6hpi

hsa−miR−92a−3phsa−miR−221−3phsa−miR−21−5phsa−miR−222−3phsa−miR−320ahsa−miR−1307−5phsa−miR−296−3phsa−miR−423−3phsa−miR−378a−3phsa−miR−671−5phsa−miR−769−5phsa−miR−320bhsa−miR−1260bhsa−miR−125b−5phsa−miR−130b−5phsa−miR−320chsa−miR−1275hsa−miR−1908−5phsa−miR−378chsa−miR−328−3phsa−miR−501−5phsa−miR−22−3phsa−miR−151a−3phsa−miR−423−5phsa−miR−186−5phsa−miR−125a−5phsa−miR−1307−3phsa−miR−181a−5phsa−miR−218−5phsa−miR−93−5phsa−miR−106b−5phsa−miR−25−3phsa−miR−26a−5phsa−miR−16−5phsa−miR−20a−5phsa−miR−19b−3phsa−miR−103a−3phsa−let−7a−5phsa−miR−196b−5phsa−miR−744−5phsa−miR−34a−5phsa−miR−101−3phsa−miR−181b−5phsa−miR−30e−5phsa−let−7e−5phsa−let−7f−5phsa−miR−15b−5phsa−miR−23b−3phsa−miR−24−3phsa−miR−18a−5phsa−miR−30d−5phsa−miR−17−5phsa−miR−19a−3phsa−miR−30a−5phsa−miR−27b−3phsa−miR−425−5phsa−miR−7−5phsa−miR−629−5phsa−miR−324−3phsa−miR−615−3phsa−miR−28−3phsa−miR−1180−3phsa−miR−339−5phsa−miR−652−3phsa−miR−181c−5phsa−miR−345−5phsa−miR−193b−3phsa−miR−1301−3phsa−miR−185−5phsa−miR−210−3phsa−miR−99b−3phsa−miR−505−3phsa−miR−452−5phsa−miR−1296−5phsa−let−7i−5phsa−let−7c−5phsa−miR−140−3phsa−miR−361−5phsa−miR−148a−3phsa−let−7g−5phsa−miR−31−5phsa−miR−17−3phsa−miR−182−5phsa−miR−26b−5phsa−miR−374a−5phsa−miR−92b−3phsa−miR−374b−5phsa−miR−130a−3phsa−miR−30c−5phsa−miR−29a−3phsa−miR−128−3phsa−miR−10a−5phsa−miR−10b−5phsa−miR−196a−5phsa−miR−23a−3phsa−miR−15a−5phsa−miR−99b−5phsa−miR−151a−5phsa−miR−224−5phsa−miR−27a−3p

10 14 18 22

SINV

Girardi et al. Fig S6

-2000

0

2000

4000

6000

8000

10000

12000

18 19 20 21 22 23 24 25 26 27 Read length (nt)

HEK293e 16hpi

-2000

0

2000

4000

6000

8000

10000

12000

18 19 20 21 22 23 24 25 26 27 Read length (nt)

Dicer-2-HEK293 16 hpi

-4000

-2000

0

2000

4000

6000

8000

10000

12000

18 19 20 21 22 23 24 25 26 27

Rea

d co

unt

Read length (nt)

DAR-HEK293 16 hpi

+ Strand - Strand

+ Strand - Strand

+ Strand - Strand

Rea

d co

unt

Rea

d co

unt

Girardi et al. Fig S7

DAR−HEK293 16hpi

SINV genomic position (nt)C

over

age

(RP

M)

−100

0

100

200

300

400

500

600

Dicer−2−HEK293 16hpi

Cov

erag

e (R

PM

)

−100

0

100

200

300

400

500

600

HEK293e 16hpi

Cov

erag

e (R

PM

)

−100

0

100

200

300

400

500

600

DAR−HEK293 6hpi

Cov

erag

e (R

PM

)

−5

0

5

10

15

20

25

30

35

Dicer−2−HEK293 6hpi

Cov

erag

e (R

PM

)

−5

0

5

10

15

20

25

30

35

HEK293e 6hpi

Cov

erag

e (R

PM

)

−5

0

5

10

15

20

25

30

35

A B

0 2000 4000 6000 8000 10000 12000

nsP1 nsP2 nsP3 nsP4 C E2 6KP

0 2000 4000 6000 8000 10000 12000

nsP1 nsP2 nsP3 nsP4 C E2 6KP

SINV genomic position (nt)0 2000 4000 6000 8000 10000 12000

nsP1 nsP2 nsP3 nsP4 C E2 6KP

0 2000 4000 6000 8000 10000 12000

NTR nsP1 nsP2 nsP3 nsP4 C E2 6KP E1

SINV genomic position (nt)0 2000 4000 6000 8000 10000 12000

nsP1 nsP2 nsP3 nsP4 C E2 6KP

0 2000 4000 6000 8000 10000 12000

nsP1 nsP2 nsP3 nsP4 C E2 6KP

SINV genomic position (nt)

SINV genomic position (nt)

SINV genomic position (nt)

NTR E1

NTR E1NTR E1

NTR E1 NTR E1

Girardi et al. Fig S8

6 hpi 12 hpi0 hpi

DNA/dsRNA

HEK293e

Dicer-2-HEK293

DAR-HEK293

Girardi et al. Fig S9

0

5

10

15

20

25

30

35

40

45

50

Poly I:C

Rat

io F

Luc

Sam

ple

/ FLu

c M

ock

HEK293e Dicer-2-HEK293 DAR-HEK293

Girardi et al. Fig S10