1

Supplementary methods and information

Comparative genomic hybridization of precipitated DNA

The karyotypes of two normal leukocytes and two MCF-7 breast cancer cells, the DNA of

which had been precipitated are compiled in Supplementary Figure 1. While the profiles of

the normal cells showed no significant deviation from the midline, the profiles of the MCF-7

cells exhibit multiple yet almost identical genomic aberrations (e.g., on chromosome 8) that

are congruent with previously published data. Therefore, the experiments with single cells of

a defined karyotype – i.e. single leukocytes and MCF-7 breast cancer cells show that cellular

DNA of a single cell can be completely precipitated.

Supplementary Figure 1. CGH profiles of two normal leukocytes (red) and two MCF-7 breast

cancer cells (blue). Genomic DNA was isolated from the supernatant after mRNA isolation.

2

The ratio profiles of normal cells lie within the dashed lines that indicate the threshold of

significance. The profiles of both cancer cells show multiple and similar chromosomal

deletions and amplifications.

3

Generation and global amplification of single cell cDNA.

The following conditions were essential to obtain sufficient sensitivity and reliability. Firstly,

tRNA and rRNA had to be removed to improve PCR amplification. Secondly, we found that

random priming requires 2000-8000-times higher concentrations of primers for cDNA

synthesis than required with previously described techniques using oligo-dT primers at 10 nM

concentrations1-3. Supplementary Fig. 2A shows that higher concentrations of random primers

for cDNA synthesis lead to increased detection rates of specific transcripts (e.g. KRAS).

Surplus primer, being an effective competitor of the subsequent tailing and amplification

reaction, had to be removed before both steps. Equally, high dNTP concentrations improved

cDNA synthesis but interfered with the subsequent tailing reaction and also needed to be

removed. Capturing of mRNA on oligo-dT coated magnetic beads provided for simple

handling during mRNA isolation and buffer exchange steps. The second primer binding site

was added by homopolymer tailing. Here, poly-dC primers binding to poly-dG tails were

found to give at least a 100-fold higher sensitivity than poly-dT primers on poly-dA tails

(Supplementary Fig. 2B, compare lanes 3, 4 to 5, 6). Tailing efficiency (in Supplementary

Fig. 2B) as well as the sensitivity of the subsequent PCR of poly-dA- and poly-dG-tailed

sequences was assessed by adding a homopolymer tail of either poly-dA or poly-dG to a

defined PCR fragment. The poly-dA- and poly-dG-tailed fragments were diluted and then

amplified by PCR with poly-dT and poly-dC primers, respectively.

The most dramatic improvement was obtained when only one primer (Supplementary

Fig. 2D) was used for global PCR instead of two (Supplementary Fig. 2C). To prove this

point we isolated, diluted, reverse-transcribed and dG-tailed mRNA of HT29 cell line cells.

The cDNA synthesis primer consisted of a 3’ random hexamer (N6) and a 5’ flanking region

4

of either a poly-dC stretch (CFL5c-N6) or a sequence of all four bases (FL4-N6), introducing

either the FL4 or poly-dC sequence at the 5’ end of the generated cDNAs. Following dG-

tailing,the cDNAs were amplified with the respective primers. Use of the FL4 primer in

combination with the poly-dC binding primers (CP2, CP3) prevented amplification,

regardless of the annealing temperature chosen (Supplementary Fig. 2C, lanes 1, 2 and 4, 5).

PCR with the CP2 primer alone resulted in amplification of a wide size range of cDNA

molecules (0.2 – 3kb; Supplementary Fig. 2D, lanes 3, 4). Even highly diluted cDNA (1:200)

was still sufficient for global amplification (Supplementary Fig. 2D, lanes 1, 2). Random

hexamers (N6), octamers (N8), alone or combined with oligo-dT (dT)15 , sharing the same

poly-dC flanking region were compared. All worked well and reliably. The best results with

single cells were obtained with a combination of poly-dC-N8 (CFL5c8) and poly-dC-(dT)15

(CFL5cT).

5

Supplementary Figure 2. Experimental conditions determining amplification success. (A)

Twenty HT29 colon carcinoma cells (lanes 1-20) were individually isolated and processed.

After cell lysis in cDNA synthesis buffer containing the detergent Igepal, groups of five single

M

80 µM 8 µM 0.8 µM 0.08 µM A

A-tail

G-tail

10-5 10-3 - B

1 2 3 4 5 6 1 2 3 4 5 CP3-FL4 CP2-FL4 1:200 pure

C D

- + 1 2 3 4 5 11 6 7 8 9 10 12 13 14 15 16 17 19 18 20

3 4 5 621

6

cells were formed and reverse transcribed with four different concentrations of random cDNA

synthesis primers. Efficacy of cDNA synthesis was tested with KRAS specific PCR for each

concentration. (lanes 1-5, 80 µM; lanes 6-10, 8µM; lanes 11-15, 0.8 µM; lanes 16-20, 0.08

µM). (B) Effect of homopolymer tails on sensitivity. A 350 bp TGFA cDNA fragment was

isolated, diluted and either dA or dG tailed. Serial dilutions were tested by PCR using poly-dT

or poly-dC containing primers, respectively, and an additional primer from within the TGFA

sequence. The informative dilutions are shown in duplicates. (lanes 1+2, negative control;

lanes 3+4, 10-3 dilution; lanes 5+6, 10-5 dilution). (C) FL4-N6 primed and reverse transcribed

mRNA was dG-tailed and amplified using the CP3 + FL4 primers (lanes 1-3) or CP2 + FL4

primers at different annealing temperatures (lane 1+4, 68°C, lane 2+5 65°C, lane 3+6,

negative control). (D) An identical amount of mRNA as in C) was reverse transcribed using

the CFL5cN6 primer, and amplified with the CP2 primer (lane 3+4), which resulted in

amplification of a wide range of cDNA fragments. Even a mRNA dilution of 1:200 (lane 1+2)

resulted in a similar smear regardless of annealing temperatures (lane 1+3, 68°C; lane 2+4,

65°C; lane 5, negative control).

Primers used and mentioned in Supplementary Fig.2C and D were:

Cfl5cN6 5’-(CCC)5 GTC TAG ANN (N)6-3’

FL4N6 5’-TTT CTC CTT AAT GTC ACA GAT CTC GAG GAT TTC (N)6-3’)

CP3 5’- GCT GAA GTG GCG AAT TCC GAT GCC (C)12-3’

FL4 5’-CTC CTT AAT GTC ACA GAT CTC GAG GAT TTC-3’.

7

Comparison of single cells of different histogenetic origin

We tested the PCR amplificates from single cells for suitability of cDNA array

analysis. The specificity of the hybridisation of digoxigenin-labelled probes is depicted in

Supplementary Table 1, where the expression patterns of genes from single cells of different

histogenetic origin are shown. Only the MCF-7 and A431 cell expressed the cytokeratin

genes, markers for their epithelial origin, whereas the erythroleukemia K562 cell and EBV-

transformed B cell JY expressed genes of a hematopoetic origin, including CD33, CD37,

CD38, and kappa light chain (IGKC) in the B cell. In addition, the testis- and tumour-specific

MAGE genes were highly expressed in all cancer cells but not the virally transformed B cell.

These data show that the amplificates obtained from single cell PCR are suitable for cDNA

array analysis and produce cell type-specific gene expression patterns of single cells.

Supplementary Table 1. Expression of histogenetically informative genes by single cells

derived from different tissues.

MCF-7 A431 K562 JY ACTB + + + + EEF1A1 + + + + KRT7 + + - - KRT10 - + - - KRT13 - + - - KRT18 + + - - KRT19 + + - - EpCAM + + - - CD33 - - + + CD37 - - + + CD38 - - + - IGKC - - - + VIM - + + -

8

ITGA6 + - - - ITGB1 + - - - ITGB2 - - - + ITGB4 - - + - ITGB7 - - - + PTK2 + - - - MageA1 + - + - MageA2 + + + - MageA3 + - + - MageA6 + - + - MageA12 + + + -

Array analysis of MCF-7 breast cancer cells

Expression patterns of four MCF-7 cells were compared using a cDNA array with 109

different genes (Supplementary Table 2). 46 genes (42%) were expressed in at least one cell,

and 63 (58%) were negative for all four cells. Eighteen of the 46 (39%) expressed genes were

detected in all four cells whereas the remaining 29 (61%) were found to be heterogeneously

expressed. All housekeeping genes (EEF1A1, GHPDH, ACTB) belonged to the genes

detected in all four cells but also structural genes like the cytokeratins (KRT) 7, 8, 18, 20 and

adhesion molecules like Integrin α6 and β1 (ITGA6, ITGB1). Among the genes that were

found only in one of the cells many are regulated within the cell cycle such as CDKN2A,

CDKN1A, CDKN1B, ING1, MKI67, TK1, but also cytokeratin family members that are

usually not expressed in breast tissue (KRT10, KRT13).

Supplementary Table 2. Commonly and differentially detected transcripts of four single

MCF-7 cells.

4/4* 3/4 2/4 1/4 EEF1A1 GHPDH ACTB

KRT19 TIMP1 CTSB

ITGB4 ITGB5 TP53

KRT10 KRT13 ADAM9

9

KRT7 KRT8 KRT18 KRT20 ITGA6 ITGB1 PTK2 BSG PLAUR MMP7 CCND1 EPHA2 M4S1 (EpCAM) ABCC1 PHLDH1

CTSD CTSL ADAM10 MYC

CKM

ADAM15 ADAM17 CDKN2A CDKN1A CDKN1B ING1 MKI67 TK1 CDH1 IGF1R IGF2R TGFB VEGF DSP

* Listed are the transcripts that were detected in all four single cells (4/4), three of four (3/4),

two of four (2/4), and one of four (1/4).

10

Primer information of the gene specific PCRs

Supplementary Table 3: Sequences of oligonucleotide primers for the RT-PCR amplification

of the different genes.

MAGE 2 sense antisense

CAT TGA AGG AGA AGA TCT GCC T CAG GCT TGC AGT GCT GAC TC

MAGE 3/6 sense antisense

GGC TCG GTG AGG AGG CAA G GAT GAC TCT GGT CAG GGC AA

MAGE 4 sense antisense

CAC CAA GGA GAA GAT CTG CCT CAG GCT TGC AGT GCT GAC TCT

β-Actin sense antisense

GTG GGG CGC CCC AGG CAC CA CTC CTT AAT GTC ACG CAC GAT TTC

EF-1α sense antisense

TGC CCC AGG ACA CAG AGA CT CTG TGT CGG GGT TGT AGC CA

TNF-α sense antisense

CAG AGG GAA GAG TTC CCC AG CCT TGG TCT GGT AGG AGA CG

EMMPRIN sense antisense

CAT GCT GGT CTG CAA GTC A GTT CTC AAT GTG TAG CTC TG

EpCAM sense antisense

GCG AGT GAG AA CTA CTG CAC ATC AGC TAT GTC CAC ATC

Ki-ras sense antisense

TAT AAA CTT GTG GTA GTT GGA GC CTC TTG ACC TGC TGT GTC GA

11

Sequence-independent amplification

In order to assess the sensitivity, the potential systematic failure to amplify “difficult”

sequences and the influence of other technical factors on transcript detection we tested the

differences between cDNA preparations of single A431 cells that were split into two halves

before global amplification. In the first step, we performed gene-specific PCRs with the

amplified cDNAs of the halved cells (Supplementary Fig. 3). For comparison, we diluted

unamplified cDNA isolated from a pool of 500,000 A431 cells to such an extent that the

intensity of the β-actin band was similar to that obtained with 50% of cDNA prepared from a

single cell. After 32 cycles and with a cDNA amount corresponding to about 10,000 cells,

both β-actin signals that of the pooled control DNA as well as that of 50% of the single cell

cDNA reached the plateau phase of amplification. As shown in Supplementary Figure 3, the

variation between two cDNA halves of the same cell was very low. In two independent

experiments, each half (a+b) from six single A431 cells yielded β-actin bands of similar

intensity.

Because PCR amplification is known to be sequence-dependent, we also tested the

amplification of MAGE transcripts, very demanding with regard to primer design. Kufer et al.

had to test almost 800 primer combinations to achieve successful amplification of MAGE

genes from less than five cells demonstrating the magnitude of the problem5. MAGE genes

belong to a family of genes expressed in spermatogonal cells and exclusively in various

cancer cells, hence the designation cancer-testis genes. MAGE transcripts could be detected in

all single cells. After the same number of cycles in the specific PCR as for β-actin, the level

of MAGE expression was consistently found to be lower than that of β-actin, and the

concentration of MAGE transcripts in split single cell samples after global PCR amplification

(Supplementary Fig. 3, lanes 2-4 and 6-8) compared well to that of the control sample from

12

pooled cells (Supplementary Fig. 3, +). Although the MAGE transcripts were only weakly

expressed in the pooled cDNA from 10,000 cells (Supplementary Fig.3, +), the data obtained

from single cells were identical in 4 out of 6 paired halves of the cDNA. The failure to detect

any MAGE transcript in cell half 7a and 8b (Supplementary Fig.3) is most likely due to an

unequal distribution of the beads prior to amplification between the two halves indicating thus

the detection limit of the method. The semi-quantitative character of our amplification method

is strongly suggested by the reproducibly similar signal intensity of all β-actin and MAGE

transcripts: No artificial increase in intensity is observed in any divided cell and the intensities

of the β-actin and MAGE band closely resemble the positive control. As the yield (y) of a

PCR amplification follows the equation y = ((1+R)n -2n)x, with n being the number of cycles,

x the copy number of the original template and (1+R) the amplification efficiency of a any

sequence (R ≤ 1), Supplementary Figure 3 suggests that R is the same for all sequences under

the applied conditions. Consequently, although an exponential amplification takes place, the

ratios of the different sequences remain very similar.

Supplementary Figure 3. Gene specific PCR for β-actin and various MAGE transcripts using

unamplified pooled cDNA of A431 cells as positive control (+) and amplificates of single

76 5321 4 8 M

Actin

Mage

Mage

Mage

M - + a b a b a b a b a b a b a b a b

13

A431 cells (lane 2-4 and 6-8) that were divided into two halves (a+b) before global PCR. Two

independent experiments were performed (lane 1-4 and 5-8) with lane 1 and lane 5 being the

negative controls for the global PCR.

14

Evaluation of array hybridization of split single cells

The high sensitivity of the procedure and the diversity of the isolated and amplified

cDNA sequences was also seen with the cDNA of four halves labeled and hybridized to a

cDNA array representing 193 different gene sequences. Most transcripts could be detected in

both halves of the single cell PCR products (Supplementary Fig.4). Of the total of 148

observed signals, 95 (64%) were found in the corresponding halves, whereas 53 (36%) were

found in only one half. Figure 4 shows the distribution of the signals of the two halves from

one experiment. Hybridization intensity of the two halves for the spotted cDNAs was

classified from 0 (no signal), 1 (weak) over 2 (middle) to 3 (strong). It can be seen that for

strong signals both halves correlated well (correlation coefficient r= 0.81). Also the signals

with intermediate strength were in most cases found in both halves. Weak signals gave the

least concordant results between the two halves of a cell. This can be explained by the

stochastic nature of the cDNA priming events, an uneven distribution of low copy number

transcripts before the PCR is performed and the undefined but real detection-limit of the

method. As many transcripts are estimated to be present in a cell in copy numbers of about 5-

15 (ref. 4), it is very likely that such transcripts are particularly subjected to effects leading to

discordant results when cDNA of a single cell is divided.

15

Supplementary Figure 4. Hybridization results of two independently amplified cDNA halves

of a single cell. Each spot on the array was classified as strong (3), middle (2), weak (1) and

no signal (0). The signal intensities for each spot were compared between the two halves. X-

and y-axis give the signal intensities for each half of the cDNA of a single cell and the z-axis

the number of cDNAs. The column at position 3/3 for example means that 10 transcripts were

expressed equally strong in both halves. Ideally, the columns would form a diagonal from 3/3

over 2/2 and 1/1 to 0. However, it can be seen that the light blue columns are of similar height

at position 0/1, 1/1, 1/0, indicating that no bipartition was achieved for weak signals as was

for the strong signals. (Red columns, at least one half produced a strong signal; yellow, at

least one half produced an intermediate signal; light blue, at least one half resulted in a weak

signal; dark blue, no signal in both halves).

16

Hybridization of exemplary micrometastatic cells to Clontech Cancer1.2 and own array

From the three micrometastatic cells C, B, L mRNA was isolated and prepared as

described. As control, the procedure was performed without the addition of a cell. Seven

genes on the Clontech arrays had to be excluded from analysis because a signal was obtained

in at least one of the negative controls. These genes were coding for VBP1, CASP10, TGFB1,

HBA1, ROS1, TDGF1, and TNFR2 . The number of positive signals ranged from 5.3 %

(70/1313), 7.0 % (92/1313) to 11.8 % (155/1313) for cells from patients B, C, and L

respectively. Although, these numbers are lower than those obtained by conventional

transcriptome analysis with millions of cells, one should not forget that the number of

attributes of an individual cell is necessarily lower than that of a population. The hybridization

results are shown in Supplementary Figure 5.

Hugo B C L Hugo B C L Hugo B C L

ABCC1 HBA1 PDGFAP

ACTB hBAP PDPK1

ACTB HDAC3 PGF

ACTB HDGF PHB

ADAM15 HINT PHKB

ADAM17 HLA PHLDA1

ADAM8 HLAC PIM1

ADPRT HLADPB1 PIN1

ADSL HLADQ1B PKUA

ADSS HLADRB1 PLA2

AHR HMG2 PLAU

AK1 HMGIY PLAUR

ANT2 HNRPK PM5

17

API4 HSPA5 PPM1G

ARAF1 IFI27 PRIM1

ARHA IFIT1 PRKACB

ARHB IFNAR1 PRKDC

ARHGDIB IFNB1 PRKM3

BAD IGF1 D123

BARK1 IGF2R PSCD2

BCGF1 IL10 PSME1

BCL7B IL12B PTB

BENE IL1B PTK2

BLMH IL1RN PTP4A1

BPGF1 IMPDH1 RAC1

BSG IMPDH2 RACK7

C5 ING1 RAD23A

CALM1 IRF1 RALB

CASP6 ITGA3 RAN

CCNA ITGAV RAP1A

CD27 ITGB2 RBBP4

CD36 ITGB3 RBL2

CD59 ITGB7 RFC2

CD74 JAG1 RFC3

CD9 JUN ROCK1

CDC25A KAI1 RPL10

CDC37 KIAA0324 RPL13A

CDC42 KRAS2 RPL32

CDC7L1 KRT10 RPL5

CDH1 KRT11 RPS16

CDK10 KRT13 RPS21

CDK5 KRT18 RPS5

CDKN1A KRT19 RPS9

18

CDKN2A KRT2E RXRB

CENPF KRT6A S100A4

CHD3 KRT7 SFRS7

CSBP1 KRT8 SFRS8

CSTB LAMC1 SGK

CTNNB LDHB SHH

CTSB LIG1 SP100

CTSD LY84L SPTB

CTSL M4S1 STI3

DDIT1 MAD STK12

DES MAGEA12 STK3

DSG2 MGMT TAPBP

DYRK4 MIC2 TDGF1

E2F1 MKI67 TGFA

EB1 MLF2 TGFB1

EEF1A1 MLH1 THBS1

EEF2 MMP7 TIEG

EGF MT1-MMP TK1

EGFR MT2-MMP TNFRSF1B

EIF2A MYBL2 TOP2A

EIF3S9 MYC TP53

EPHA2 NCK1 TRAM

ERCC1 NEDD5 TRAP1

ETV4 NFKBIA TRIP

FAU NME1 TUBA1

FBP3 NME2 TYMS

FGF6 NME3 UB

FRA1 NME4 UBL1

G1P3 NOTCH1 UMK

GHPDH NTHL1 UMPS

19

GLG1 OSMR VEGF

GMFB PA2G4 VIM

GRP58 PAK2 VRK2

GST PCTK1

GUK1 PCTK2

HAI2 PCTK3

PDGFA

Supplementary Figure 5. Hybridization results of cell B, C, and L. Three categories of signal intensity were formed: Strong (black), middle (middle gray) and weak (light gray).

20

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