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TRANSCRIPT
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
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
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