supporting information - pnas...2cl 2), meoh, and n,n′-dimethylformamide, and subsequently mixed...

Supporting InformationArango et al. 10.1073/pnas.1303726110SI Materials and MethodsPreparation of Apigenin-Immobilized Polyethyleneglycol-PolyacrylamideBeads.Apigenin was immobilized to amino polyethyleneglycol-polyacrylamide copolymer beads (PEGA beads; EMD Bio-sciences). Briefly, 0.40 mmol/mg PEGA beads were washedthree times with pyridine and subsequently mixed with 3.3 molequivalents (to the amino group loaded on the beads) of4-nitrophenyl bromoacetate. Beads were stirred at room temper-ature for 3 h, filtered, washed three times with ∼10 mL each di-chloromethane (CH2Cl2), MeOH, and N,N′-dimethylformamide,and subsequently mixed with 2.2 mol equivalents apigenin and1.8 mol equivalents K2CO3 to the bromoacetyl group loaded onthe beads. The resulting suspension was stirred at room tem-perature for 3 d, filtered, and washed three times with ∼10 mLeach CH2Cl2, MeOH, and H2O. The apigenin-immobilized PEGAbeads were vacuum-dried. Filtrates were neutralized by the ad-dition of 1 M aqueous HCl. After layer separation, the aqueouslayer was extracted seven times with 50 mL EtOAc. The com-bined organic layer was washed with brine, dried over anhydroussodium sulfate (Na2SO4), and concentrated in vacuo. The un-loaded apigenin was recovered from the supernatant by silicagel chromatography (chloroform/MeOH = 20:1). The amountof apigenin immobilized on 1 mg dried PEGA beads was es-timated to be ∼0.14 μmol as determined by subtracting theamount of unloaded apigenin from the amount of apigenin usedin the reaction.

Plasmid Construction. pEGFP–heterogeneous nuclear ribonu-cleoprotein A2 (hnRNPA2) and plasmid T7-driven expression 9c(pET9c)-hnRNPA2 were provided by Lexie Friend (Universityof Queensland, St. Lucia, Australia) (1) and Adrian R. Krainer(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) (2),respectively. pEGFP-BAG1 (B-cell lymphoma 2-associatedathanogene 1) L and pEGFP–Rho-guanine nucleotide ex-change factor 1 were obtained from Ann C. Williams (BristolUniversity, Bristol, United Kingdom) (3) and Philip B. Wede-gaertner (Kimmel Cancer Institute, Philadelphia) (4), respectively.The different hnRNPA2 cDNA fragments were amplified usingthe pET9c-hnRNPA2 clone as a template and cloned into a plas-mid entry-driven topoisomearse (pENTR-D-TOPO) vector (LifeTechnologies). The following primers were used to generate the

PCR products comprising different fragments of hnRNPA2 (Ta-ble S1): to generate hnRNPA2 full length, PrimersAndreaOhio(PAO)-351 and PAO-338; for hnRNPA2ΔC-terminal region(hnRNPA2δC, amino acids 1-263), PAO-351 and PAO-337; forhnRNPA2δglycine-rich domain (hnRNPA2δGRD, amino acids 1–189), PAO-351 and PAO-372; for hnRNPA2glycine-rich domain(hnRNPA2GRD, amino acids 190–341), PAO-374 and PAO-338;and for hnRNPA2C-terminal region (hnRNPA2C, amino acids264–341), PAO-352 and PAO-338. To generate the GST-taggedand 6xHis-tagged fragments, the pENTR-D-TOPO containinghnRNPA2 fragments was cloned into pDEST15 and pDEST17vectors, respectively, by recombination using the Gateway LRClonase Enzyme Mix and the conditions suggested by the manu-facturer (Life Technologies). To generate the fluorescent in-dicator protein (FLIP) constructs, hnRNP-A2C cDNA wasamplified by PCR from the pET9c-hnRNPA2 vector using thePAO-379 and PAO-378 primers pair (Table S1). Fragments werecloned into the pENTR-D-TOPO vector and transferred to the6xHis-tagged pFLIP vectors by recombination using the GatewayLR Clonase Enzyme Mix. pFLIP vectors, previously used togenerate biosensors, were generously provided by Wolf Frommer(Carnegie Institution for Science, Stanford, CA) (5). ThehnRNPA2C fragment was cloned into pFLIP1 or pFLIP2 vectorscontaining the N-terminal CFP and C-terminal YFP tags orpFLIP4 vector containing the N-terminal GFP and C-terminalmonomeric Kusabira-Orange. pFLIP2 and pFLIP4 vectors holdthe Gateway recombination linkers flanked by the KpnI and SpeIrestriction sites (Fig. S5). Different versions of FLIP2-hnRNPA2C

(referred as pFLIP2-1, -2-2 and -2-3-hnRNPA2C) and FLIP4-hnRNPA2C (referred as pFLIP4-1 and -4-2-hnRNPA2C) weregenerated by digestion and self-ligation using KpnI and/or SpeIsites to improve FRET emission (Fig. S5).

Cell Culture. MDA-MB-231 breast cancer cells were grown inDMEM supplemented with 10% (vol/vol) FBS and 1% penicillin/streptomycin. MCF-10A immortalized breast epithelial cells weregrown in DMEM/F12 medium supplemented with 10% (vol/vol)FBS, 1% penicillin/streptomycin, 20 ng/mL EGF (Prepotech),0.5 mg/mL hydrocortisone (Sigma), 100 ng/mL cholera toxin(Sigma), and 10 μg/mL insulin (Sigma). All cells were obtainedfrom ATCC and cultured at 37 °C in 5% CO2 environment.

1. Friend LR, Han SP, Rothnagel JA, Smith R (2008) Differential subnuclear localisation ofhnRNPs A/B is dependent on transcription and cell cycle stage. Biochim Biophys Acta1783(10):1972–1980.

2. Mayeda A, Munroe SH, Cáceres JF, Krainer AR (1994) Function of conserved domains ofhnRNP A1 and other hnRNP A/B proteins. EMBO J 13(22):5483–5495.

3. Barnes JD, et al. (2005) Nuclear BAG-1 expression inhibits apoptosis in colorectaladenoma-derived epithelial cells. Apoptosis 10(2):301–311.

4. Bhattacharyya R, Wedegaertner PB (2003) Characterization of G alpha 13-dependentplasma membrane recruitment of p115RhoGEF. Biochem J 371(Pt 3):709–720.

5. Fehr M, Frommer WB, Lalonde S (2002) Visualization of maltose uptake in living yeastcells by fluorescent nanosensors. Proc Natl Acad Sci USA 99(15):9846–9851.

Arango et al. www.pnas.org/cgi/content/short/1303726110 1 of 9

www.pnas.org/cgi/content/short/1303726110

HO

H

HO

H

NaringeninApigenin

A

B

C A

B

C

1

23

456

7

8 1’2’

3’4’

5’6’

Filtered reads (0.2~1.7M)

Align to human coding sequences

OHO

O

O

OHO

O

O

Gene A Gene B

1 0.5 0.5

ICPG=1.5 ICPG=0.5

nICPGA = ICPGA ICPG( ) 106

A

B

Fig. S1. Chemical structure of flavonoids and analysis of identified sequences to determine putative apigenin target enrichment. (A) Schematic representationof chemical structures of the flavone apigenin and the flavanone naringenin. (B) Calculation of normalized in frame-aligned counts per gene (nICPG) model.Sequences were filtered and aligned to human coding sequences. The number of sequences aligned in frame to a single gene was considered as the in frame-aligned count (blue bar), whereas alignment of a single sequence to multiple genes was considered as the weighted count (purple bar).



C-E

2

A-E

2

C-E

1

A-E

1

Inpu

t1

Inpu

t2

Ori-

lib

4.0

1.0

2.0

3.0

0

log1

0(n

ICP

G)

4.0

1.0

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A-E

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C-E

1

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1

Inpu

t1

Inpu

t2

Ori-

lib

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t1

Inpu

t2

Ori-

lib4.0

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C-E

2A

-E2

C-E

1A

-E1

Inpu

t2

log10(nICPG)

Inpu

t1O

ri-lib

15,5

68 g

enes

IV

III

III

C-E

2

A-E

2

C-E

1

A-E

1

Inpu

t1

Inpu

t2

Ori-

lib

4.0

1.0

2.0

3.0

0

Signal distribution

Cluster I

Cluster II

Cluster III

Cluster IV

GTPase activationMembrane

Alternative splicingIon transport

PhosphoproteinThyroid hormones biosynthesis

Calcium transportDNA damage

Transport1 0.1 0.01 0.001

p-value

Host-virus interactionTandem repeat

CentromereCell cycle

Tumore suppressorBlocked amino end

IntestineIron

Pyroglutamic acidTransmembrane protein

GonadHeme

Vitamin B12 transportCobalt transport

Paired boxSignal-anchor

Enriched functional categories

Cluster II

Cluster III

Cluster IV

CBA

Cluster I

1 0.1 0.01 0.001p-value

1 0.1 0.01 0.001p-value

1 0.1 0.01 0.001p-value

log1

0(n

ICP

G)

log 1

0(n

ICP

G)

log1

0(n

ICP

G)

Fig. S2. Hierarchical clustering analysis of the phage display coupled with second generation sequencing (PD-Seq) results. (A) Heat map representation oflog10(nICPG) for the minimal 15,568 genes present in the phage display library analyzed by hierarchical clustering using the average linkage method. Thecolumns in the heat map correspond to the columns described in Fig. 1C. An enlarged view of cluster I is shown in Fig. 1E. Selected clusters are indicated byRoman numbers. (B) Line graph indicating distribution of log10(nICPG) within clusters. The red line indicates log10(nICPG) of hnRNPA2/B1. (C) The bar graphdisplays functional categories enriched in each cluster, and the x axis (log scale) indicates the P value. Filled black bars correspond to functional categoriesenriched significantly (P < 0.01).



01

A-beads

bio-panning

6

2

4

C-beads

φx 1

0-3

(E

lute

/Inpu

t)

st 2nd 3rd

Inpu

t1

C-E

1

A-E

1

Inpu

t2

C-E

2

A-E

2

Inpu

t3

C-E

3

A-E

3

A B

1 2 3 4 5 6 7 8 9

500 bp

200 bp

hnRNPA2MKET

A-E3-2 PNSSGNQGGGYGGGYDNYGGGNYGSGNYNDFGNYNQQPSNYGPMKSGNFGGSRNMGGPYGGGNYGPGGSGGSGGYGGRSRY*

A-E3-5 PNSMKETAAAKFERQHMDSPKLAAALE*

A-E3-6 PNSSGNQGGGYGGGYDNYGGGNYGSGNYNDFGNYNQQPSNYGPMKSGNFGGSRNMGGPYGGGNYGPGGSGGSGGYGGRSRY*

A-E3-16 PNS

C-E3-1 PNSMKETAAAKFERQHMDSPKLAAALE*

C-E3-16 PNSMKETAAAKFERQHMDSPKLAAALE*

C Phage

Red: hnRNPA2/B1Black: MKET peptide

Total reads MCS reads* MKET reads

Original library 4,859,548 1,014,536 2,971,167Input1 7,817,706 1,438,079 4,640,635C-E1 7,318,918 1,637,884 3,976,456A-E1 5,755,257 1,231,065 2,802,825

Input2 3,863,523 769,109 2,816,430C-E2 6,250,021 1,485,813 4,153,495A-E2 10,694,740 1,871,830 7,407,376Total 46,559,713 9,448,316 28,768,384

D

* MCS: Multicloning site (no insert)

EcoRI HindIII

ATG CTC GGG GAT CCG AAT TCT CCT GCA GGG ATA TCC CGG GAG CTC GTC GAC AAG CTT GCC GCG GCC GCA CTC GAG TAA

EcoRI HindIII

Left arm Right arm

SacI SalISmaIEcoRV

Left arm Right armATG CTC GGG GAT CCG AAT TCG ATG AAA GAA ACC GCT GCT GCT AAA TTC GAA CGC CAG CAC ATG GAC AGC CCA AAG CTT GCG GCC GCA CTC GAG TAA*

PNSMKETAAAKFERQHMDSPKLAAALE*

DNA sequence of empty vector

DNA sequence of MKET clone

Peptide sequence of MKET clone Left arm Right arm

azorectase (Pseudomonas aeruginosa)1-MKETAAAKFERQHMDSP-17

azorectase (Brevibacillus laterosporus)repetitive protein BP68 (synthetic construct)mutant alpha-amylase (synthetic construct)

kinesin tail 864 (Expression vector p864INS)

Expression vector pet22-srt A

S.Tag peptide (Plasmid pPV)

14-MKETAAAKFERQHMDSP-3018-MKETAAAKFERQHMDSP-3418-MKETAAAKFERQHMDSP-3418-MKETAAAKFERQHMDSP-3418-MKETAAAKFERQHMDSP-346-MKETAAAKFERQHMDSP-22

MKET clone 1-MKETAAAKFERQHMDSP-17 ATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCA

1-248-57-41-52-52-

5127-1717-

-51-298-107-91-102-102-5177-1767

aminoacid alingment nucleotide alingmentF

E

Insert

SGNQGGGYGGGYDNYGGGNYGSGNYNDFGNYNQQPSNYGPMKSGNFGGSRNMGGPYGGGNYGPGGSGGSGGYGGRSRY*

Fig. S3. Conventional phage display identifies clones that bind to the apigenin-loaded beads (A-beads) and are highly enriched in the PD-Seq. (A) Phage (ϕ)enrichment was determined by counting pfu per milliliter in elutions from A- (white bars) and unloaded control beads (C-beads; black bars) after each round ofbiopanning (first, second, and third). (B) Enrichment of selected clones was determined by PCR using T7 primers (Table S1) flanking the insert after every roundof biopanning. (C) Peptide sequences of apigenin binding peptides isolated by conventional phage display. Sequences in red correspond to the hnRNPA2/B1peptide fragment, and sequences in black correspond to the MKET noncoding fragment. (D) Summary of sequence reads, total number of reads, multicloningsite (MCS) reads, and MKET reads for each of the libraries generated and sequenced by Illumina GAII. (E) Nucleotide sequence of the phage MCS without insertor containing the MKET clone. (F) Alignment of the MKET DNA and peptide sequences indicating its probable origin.



φ h

nRN

PA2

bi

ndin

g %

100

75

50

25

0DMSO Apigenin Naringenin

* c

125

1 2 3 4

C A C A

ARHGEF1-GFP

148

98

64

50

kDa C A C A

GFP-BAG1

148

98

64

50

kDa

0

0.2

0.4

0.6

0.8

0 10 20 30 40

DMSO Apigenin Naringenin Inhibitor

Time (min)

IDH

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tivity

(uni

ts/m

g)

0 10 20 30 40Time (min)

UG

DH

act

ivity

(uni

ts/m

g)

0

0.1

0.2

0.3

0.4 DMSO Apigenin Naringenin Inhibitor

Bound Supernatants

1 2 3 4

Bound Supernatants

A B

C D E

**

*

*

Fig. S4. Validation of apigenin targets. (A) A-beads were coincubated with selected phages in the presence of 20 μM apigenin, naringenin, or DMSO. Relativebinding percentage to A-beads was determined by counting pfu per milliliter ϕ-hnRNPA2C relative to DMSO control. Data represent the mean ± SEM (n = 3).*P < 0.05. (B and C) MDA-MB-231 cells were treated with 50 μM apigenin, naringenin, or DMSO for 3 h. (B) Isocitrate dehydrogenases 3 (IDH3) and (C) UDP-glucose dehydrogenase (UGDH) activities were measured in mitochondria and whole-cells lysates, respectively, as described in Materials and Methods. Io-doacetic acid (1 M), an enzymatic inhibitor, was added as control. Data represent mean ± SEM (n = 4). *P < 0.05 determined by two-way ANOVA. (D and E)Lysates from HeLa cell expressing full-length (D) Rho-guanine nucleotide exchange factor 1-GFP or (E) GFP-BCL2-associated athanogene 1 (GFP-BAG1) wereused in pull-down assays with A- or C-beads (indicated as A or C, respectively), resolved by SDS/PAGE, and analyzed by Western blot using anti-GFP antibodies.



0

400

pFLIP2-11200

470 500 530 560 590

800

00.10.5151025

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2000

pFLIP2-26000

470 500 530 560 590

4000

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45 nt 54 nt

CFP YFP hnRNPA2C pFLIP1-hnRNPA2C

45 nt 54 nt

CFP YFP hnRNPA2C K

pnI

Kpn

I

SpeI

SpeI

GGTACC GGTACCGGTACCGGTACC

GCTAGC GCTAGCGCTAGC GCTAGC

pFLIP2-hnRNPA2C

CFP YFP

CFP YFP

pFLIP2-1-hnRNPA2

pFLIP2-2-hnRNPA2

C

C

hnRNPA2C

hnRNPA2C

pFLIP4-hnRNPA2C

45 nt 54 nt

GFP mKO

Kpn

I K

pnI

SpeI

SpeI

GGTACC GGTACCGGTACCGGTACC

GCTAGC GCTAGCGCTAGC GCTAGC

pFLIP4-1-hnRNPA2 C GFP mKO

SpeI

SpeI

pFLIP4-2-hnRNPA2 C GFP mKO

Kpn

I K

pnI

hnRNPA2C

hnRNPA2C

hnRNPA2C

oitar PFC/ PFYWavelength (nM) Apigenin (μM)

oitar PFC/ PFY

Wavelength (nM) Apigenin (μM)

Wavelength (nM)

C His

C His

His

His

His

His

His

RFU

RFU

RFU

pFLIP2-1

pFLIP2-2

pFLIP1

pFLIP2

Emission Spectrum YFP/CFP Ratio

Apigenin (μM)

Fig. S5. Development of a flavonoid nanosensor. Bacteria lysates expressing different versions of FLIP-hnRNPA2C proteins were incubated with increasingconcentrations of apigenin (0, 0.01, 0.1, 1, 5, 10, and 25 μM) for 3 h at 37 °C. Relative fluorescence units (RFUs) were determined by spectrofluorometry (λext =405 nm; λemi = 460–600 nm) and represented as emission spectra. The calculated YFP/CFP fluorescent ratios (530/480 nm) are also represented over the 0- to25-μM concentration range of apigenin.



Apigenin Naringenin

0

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1.2

285 325 365 405 445

Abs

orba

nce

Wavelength 485 525

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40

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015102550100

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1000

1500

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RFU

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600

460 500 540 580

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RFU

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RFU

Wavelength

Flavonoid (μM)

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600

460 500 540 580

RFU

Wavelength

Naringenin Apigenin

0

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5000

510 530 550 570 5900

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OHO

OHO

HO

CFP YFPCFP YFP

hnRNPA2C hnRNPA2C

RFU

RFU

Wavelength Wavelength

A

B

C

D

E

015102550100

Flavonoid (μM)

015102550100

Flavonoid (μM)

Exc

itatio

n

405 nm 480 nm 530 nm 405 nm 480 nm 530 nm

OH

O

O

HO

HO

Fig. S6. Effect of flavonoids on CFP and YFP fluorescence. (A) Schematic representation of the flavonoid nanosensor. (B) Apigenin absorption spectra partiallyoverlap with the CFP excitation spectrum. Absorption spectra of 100 μM apigenin and naringenin were measured using a spectrofluorometer plate reader overthe 285- to 475-nm range. The CFP and YFP excitation wavelengths were obtained from the literature (1). (C) The YFP excitation/emission spectrum of FLIP-hnRNPA2C was not affected by apigenin. FLIP-hnRNPA2C was incubated with increasing concentrations (0, 1, 5, 10, 25, 50, and 100 μM) of apigenin or nar-ingenin. RFUs were determined by spectrofluorometry and represented as emission spectra. (D) Apigenin quenches CFP fluorescence. Purified (D) CFP or (E) YFPwas incubated with increasing concentrations (0, 1, 5, 10, 25, 50, and 100 μM) of apigenin or naringenin. RFUs were determined by spectrofluorometry andrepresented as emission spectra. CFP λext = 405 nm; CFP λemi = 460–600 nm; YFP λext = 475 nm; YFP λemi: 510–600 nm.

1. Patterson G, Day RN, Piston D (2001) Fluorescent protein spectra. J Cell Sci 114(Pt 5):837–838.



0

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CFP

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Wavelength (nM) Flavonoid (μM)

Naringenin Naringenin

Eriodictyol Eriodictyol

Quercetin

Kaempferol Kaempferol

1200

OHO

O

HO

HO

OHO

OHO

HO

HO

OHO

OHO

HO

HO

HO

OHO

OHO

OH

HO

470 500 530 560 590

470 500 530 560 590

470 500 530 560 590

470 500 530 560 590

0

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OHO

Ogluc-O

HO

470 500 530 560 590

0

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1200

470 500 530 560 590

Luteolin Luteolin

OHO

OHO

HO

HO

0

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OHO

OHO

gluc

470 500 530 560 590

HO

0

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800

1200

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Flavopiridol Flavopiridol

OHO

OHO

Cl

CH3N

HO

0

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OHO

O

HO

HO

Genistein Genistein

0 1 5 10 25 10050Flavonoid (μM)

470 500 530 560 5900

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OHO

HO

CHO 3

Chrysoeriol Chrysoeriol

800

A D

EB

FC

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2.2

0 20 40 60 80 100

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2.2

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2.2

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Quercetin

1.6

1.8

2

2.2

0 20 40 60 80 100

1.6

1.8

2

2.2

0 20 40 60 80 100

Emission Spectrum YFP/CFP Ratio Emission Spectrum YFP/CFP Ratio

RFU

YFP/

CFP

ratio

Wavelength (nM) Flavonoid (μM)

Apigenin 7-O-glucoside

Apigenin 6-C-glucoside O

Fig. S7. Flavonoid structural relationship provided by the FRET-based flavonoid nanosensor. The affinity-purified FLIP2-3-hnRNPA2C protein was incubatedwith increasing concentrations of the indicated flavonoids (0, 1, 5, 10, 25, 50, and 100 μM) for 3 h at 37 °C. RFUs were determined by spectrofluorometry (λext =405 nm; λemi = 460–600 nm) and represented as emission spectra. The calculated YFP/CFP fluorescent ratios (530/480 nm) are also represented over the 0- to100-μM concentration range for each flavonoid. The chemical structures of the corresponding flavonoids are shown. Data represent the mean ± SEM (n = 3).(A) Flavones: luteolin and chrysoeriol. (B) Apigenin glucosides: apigenin 7-O glucoside and apigenin 6-C glucoside. (C) Flavopiridol. (D) Flavanones: naringeninand eriodictyol. (E) Flavonols: quercetin and kaempferol. (F) Genistein. Statistical significance of the variation of the observed YFP/CFP ratios over the testedflavonoid concentration range was conducted by one-way ANOVA. Black curves represent P < 0.05, and broken gray curves represent P ≥ 0.05.



1 2

DMSO Api

MDA-MB-231B

A

GAPDH

3 4

DMSO Api

MCF-10A

hnRNPA2

0

0.2

0.4

0.6

0.8

1.0

DMSO Api

MDA-MB-231

MCF-10A

Rel

ativ

e m

RN

A e

xpre

ssio

n hnRNPA2

Fig. S8. Expression of hnRNPA2 in breast epithelial cells. MDA-MB-231 breast cancer epithelial cells and MCF-10A noncarcinogenic epithelial breast cells weretreated with 50 μM apigenin or diluent DMSO control for 48 h. (A) hnRNPA2 expression was evaluated by quantitative RT-PCR. Data represents mean ± SEM(n = 4). (B) hnRNPA2 expression level determined by Western blots. Data are representative of three independent blots.

Table S1. Primers used to generate Illumina libraries, hnRNPA2 clones, and analysis of splicing

Primer Sequence Forward/reverse

PAO-351 5′-AAGGAAAAAAGCGGCCGCCATGGAGAGAGAAAAG-3′ ForwardPAO-338 5′-TTATAGGCGCGCCCGTATCGGCTCCTCCCA-3′ ReversePAO-337 5′-AAGGCGCGCCCATATCCAGGTCCTCCACCA-3′ ReversePAO-372 5′-AAGGCGCGCCCAGAACTCTGAACTTCCTGC-3′ ReversePAO-374 5′-AAGGAAAAAAGCGGCCGCCGGAAGAGGAGGCAAC-3′ ForwardPAO-352 5′-AAGGAAAAAAGCGGCCGCCGGCAACCAGGGTG-3′ ForwardPAO-377 5′-AAGGAAAAAAGCGGCCGCCGGTACCATGGAGAGAGAAAA-3′ ForwardPAO-379 5′-TTATAGGCGCGCCCACTAGTGTATCGGCTCCTC-3′ ReversePAO-378 5′-AAGGAAAAAAGCGGCCGCCGGTACCGGCAACCAGGGTG-3′ ForwardPAO-462 5′-AGACCAGTGGACATTGGTTC-3′ ForwardPAO-463 5′-GGTCCCTCCAGGAAACAAA-3′ ReversePAO-545 5′-CTTGGCCAATTTGCCTGTAT-3′ ForwardPAO-546 5′-GGCAGAAACTCTGCTGTTCC-3′ ReversePAO-547 5′-CGAGGCAAGATAAGCAAGGA-3′ ForwardPAO-548 5′-CACATGGAACAATTTCCAAGAA-3′ ReversePAO-673 5′-GGACCACCGCATCTCTACAT-3′ ForwardPAO-674 5′-TCTCCGCAGTTTCCTCAAAT-3′ ReversePAO-230 5′-ACTTTGGTATCGTGGAAGGACT-3′ ForwardPAO-231 5′-GTAGAGGCAGGGATGATGTTCT-3′ ReverseT7 insert up 5′-NNNATGCTCGGGGATCCGAATT-3′ ForwardT7 insert down 5′-NNNAACCCCTCAAGACCCGTTTAG-3′ Reverse

Table S2. Summary of reads obtained by PD-Seq

Library Total reads Filtered reads Aligned reads In-frame reads

Original library 4,859,548 873,845 661,351 545,038Input1 7,817,706 1,738,992 1,332,165 1,102,007C-E1 7,318,918 1,704,578 1,135,646 761,372A-E1 5,755,257 1,721,367 1,149,144 751,676Input2 3,863,523 277,984 208,054 172,963C-E2 6,250,021 610,713 320,373 176,654A-E2 10,694,740 1,401,628 508,012 313,289Total 46,559,713 8,329,107 5,314,745 3,822,999

Filtered reads correspond to the number of reads minus the MKET clonecontaminant and reads with no insert (MCS reads) (Fig. S3).



supporting information - pnas...2cl 2), meoh, and n,n′-dimethylformamide, and subsequently mixed...

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