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Supplementary Material and Methods Long distance inverse PCR The tumor DNA was digested at 37°C for 2 hours with the restriction enzymes SphI, MfeI, XbaI (Fermentas, St. Leon-Rot, Germany) followed by heat-inactivation at 68°C for 30 minutes. To circularize the resulting DNA fragments, 40 ng of the digested DNA were ligated at 4°C overnight in a total volume of 50 µL with 2 U of T4 DNA ligase (Invitrogen, Groningen, Netherlands). The ligated DNA was then isopropanol precipitated and dissolved in a final volume of 10 µl Tris. The oligonucleotides in the first round of amplification were: MfeI ex: 5’-GCTCAGCTAAGAAATACTAGCACAGTG-3’, SphI ex: 5’- CTTTAGAATTATGAGGTGCGCTGTGTG-3’, XbaI ex: 5’- CCTTGTCTAAGTCATTGACTGTAGGTC-3’, JH6 ex: 5’- TGCAATGCTCAGGAAACCCCACAGG-3’. PCR reaction mixture (30 µL total volume) contained 9 µL of 3.3x Gene Amp XL Buffer II (Applied Biosystems, Foster City, CA, USA), 200 µmol/L dNTPs, each primer at 0.2 µmol/L, 0.4 U rTth polymerase (Applied Biosystems), 1.25 mmol/L Mg(OAc)2 and 10 µL of the ligated DNA. Cycling conditions for the first round of PCR were 4 min at 95°C, 34 cycles of 45 s at 95°C, 45 s at 66°C and 8 min at 68°C, followed by a final extension step of 15 min at 72°C. Nested amplification with the internal primers (MfeI int: 5’-GCTGGAAGCAGATGATGAATTAGAGTC-3’, SphI int: 5’AGAGAACTGTCGGAGTGGGTGAATC-3’, XbaI int: 5’- ACCCTTGAAAGTAGCCCATGCCTTC-3’, JH6 int: 5’- TAGCAGAAAACAAAGGCCCTAGAGTG-3’) was performed for 45 cycles using identical conditions. The amplificates were purified by agarose gel electrophoresis following standard procedures and bidirectionally sequenced with the oligonucleotides used for amplification, using the BigDye kit (Applied Biosystems) and an automated sequencing apparatus (ABI3100, Applied Biosystems). Sequences were compared to published sequences using the BLAST tool (www.ncbi.nlm.nih.gov/BLAST). The presence of the chromosomal 1 translocations amplified from NHL in microdissected HRS cells was verified using the following primers in semi-nested PCRs together with the JH6 int primer: MCRext 5´and CCCCATTGAGGTAGGTGATTATGT-3´ TTTGGTTTCATGCGAACTAACAAAG-3 for GGCCACAGTCCTATCAGACTAGAGC-3´ case 1, and ATCCAGATGGCAAATGACCAGCAG-3´ ATCTATGGTGGTTTGACCTTTAGAG-3´ MCRint for case 2 and and MBRext 5´5´- MBRint 5´- CoSWCHR11.1 5´- CoSW11.2 5´- GATGACCAACCACCTGTCCAGACC-3´ for case 5. All reactions were carried out in a two rounded PCR with the following cycling conditions: 180 s at 95°C, 35 cycles for the first or 45 cycles for the second round of PCR consisting of 30 s at 95°C, 60 s at 61°C, 45 s at 72°C and a final extension of 180 s at 72°C. Analysis of allele-specific expression of BCL2 in FL/HL composite lymphomas To screen for monoallelic single nucleotide polymorphisms (SNPs), which would allow us to discriminate the translocated and the non-translocated BCL2 alleles in cases 1 and 2 we first sequenced the coding regions of the BCL2 gene in cases 1 and 2 using genomic DNA from whole tissue sections. In case 1 we found one SNP whereas no SNP was detected in case 2. We designed specific primers to detect the SNP in case 1 in a semi-nested RT-PCR (BCL2798-Pol-US 5´-CAGGATATTTAATGACAACCTTCTGG-3´, BCL2-798-Pol-LS1 5´GAATGATTCACTGGGTAAGACTAAAGG-3´, BCL2-798-Pol-LS2 5´ACTCTCCAATTCAGTTTCACATTGC-3´). Since the breakpoint in this case is located approx. 30 kb downstream of the BCL2 coding sequence it was not possible to amplify the breakpoint and polymorphic position in a single PCR product. RNA was extracted from 300 laser microdissected tumor cells of each FL and HL using the Purescript kit following the recommendations of the manufacturer (Gentra, Minneapolis, MM, USA). Each sample was divided into two parts and a cDNA synthesis was 2 performed in parallel with a control reaction without addition of reverse transcriptase (-RT control) using a Roche 1st strand cDNA synthesis kit (Roche, Mannheim, Germany). cDNA was diluted to single molecule level and a PCR with the SNP-specific primers was performed. –RT control reactions were used as negative control, which were negative for all 16 analyzed FL samples. However, in 2 of 24 negative controls from the HL samples the product from the translocated allele was amplified. Because the PCR was not intron spanning, we assume that these products are DNA contamination deriving from incomplete DNAse digestion. Cycling conditions for the first round of PCR were 180 s at 95°C, 35 cycles of 30 s at 95°C, 60 s at 61°C and 45 s at 72°C, followed by a final extension step of 180 s at 72°C. In the second round of PCR 45 instead of 35 cycles were performed. All other conditions remained unchanged. The amplicons were purified by agarose gel electrophoresis and bidirectionally sequenced with the oligonucleotides used for amplification. Analysis of the ATM gene Mutation analysis of the ATM gene was performed by denaturing high performance liquid chromatography (dHPLC) using genomic DNA of the corresponding NHLs. Briefly, PCR was carried out for each of 62 coding exons and flanking intronic sequences using 60 primer pairs modified from a previous protocol.(1) Reactions contained 50 ng genomic DNA, 0.2 µmol/L dNTPs, 0.2 µmol/L each primer, 1.5 mmol/L MgSO4, 1 U Optimase (Transgenomic, Omaha, NE, USA). Cycling conditions were 5 minutes at 95°C, followed by 40 cycles of 30 s at 95°C, 30 s at 53°-58˚C and 60 s at 72°C, and finally 7 minutes at 72°C. PCR products were heated to 95°C for 5 minutes then cooled slowly to 65°C, to allow formation of any potential heteroduplexes. Reaction products were analysed by dHPLC using the WAVE® DNA fragment analysis system (Transgenomic). The temperature for analysis for each exon was predicted using Wavemaker software (Transgenomic). Chromatograms were analysed visually and the PCR product was sequenced if a variant pattern was detected. 3 Mutational analysis of the tumor suppressor genes TP53, NFKBIA and FAS For single or few cell PCR analysis samples were incubated with 0.5 mg/mL proteinase K (Roche) for 4 h at 55°C and then 10 min at 96°C. Exons 3-6 of the NFKBIA gene were amplified by PCR from genomic DNA as described.(2) Exons 1 and 2 of NFKBIA were amplified in the first round of PCR using the oligonucleotides: 1a: 5’- GCCGACGACCCCAATTCAAAT C-3’, 1c: 5’-CATCGCTGGTCCCCCGGCTC-3’, 2d: 5’GCTACGTCCCAGGGTCAGAGAG-3’, 2a2: 5’-TGGTGGATGGCAGCGTCGCCACAC3’. Semi-nested amplification of each exon was performed using 1a, 1r: 5’GCGTCCCGCCCTCCCGACGA-3’, 2d, 2b2: 5’-GCCTGCCAGGAACACTCAGCTC-3’. PCR conditions were as described,(2) with the modification that the reactions were supplemented with 1.5x PCR Enhancer (Invitrogen). Exon 9 of the FAS gene was amplified from genomic DNA according to a published protocol.(3) Exons 7 and 8 of the FAS gene were amplified using the oligonucleotides: E7F: 5’-CTTCTTATATTTCTCTTAGTGTG-3’, E7R: 5’-CTCCATCTCAAACAAAATGAAAC-3’, TTAAGGAAAAATTAGAAGTTCACA-3’, E8F: E8R: 5’5’- GTAGAATGTATGAGAAATAACAAG-3’ in the first round of amplification. Reaction mixes were supplemented with 0.125 µmol/L of each primer and 3 mmol/L MgCl 2 for exon 8 and 2 mmol/L MgCl2 for exons 7 and 9. After incubation for 5 min at 95°C, 35 cycles of 50 s at 95°C, 30 s at 55°C, and 60 s at 72°C were followed by 10 min at 72°C. Nested amplification of the individual exons was performed using identical conditions with internal primers (E7Fi: 5’-TATGTTCTCACATGCATTCTAC-3’, AAGTAACAAAAAGCCAAATCAC-3’, E8Fi: E7Ri: 5’- 5’-TTTATTTGTCTTTCTCTGCTTC-3’, E8Ri: 5’-CCATAATATGTCACTGAAATTAG-3’). 4 References for supplementary Material and Methods 1. 2. 3. Noppe SM, Heirman C, Bakkus MH, Brissinck J, Schots R, Thielemans K. The genetic variability of the VH genes in follicular lymphoma: the impact of the hypermutation mechanism. Br J Haematol 1999;107:625-640. Jungnickel B, Staratschek-Jox A, Bräuninger A, Spieker T, Wolf J, Diehl V, Hansmann ML, Rajewsky K, Küppers R. Clonal deleterious mutations in the IkappaBalpha gene in the malignant cells in Hodgkin's lymphoma. J Exp Med 2000;191:395-402. Müschen M, Re D, Bräuninger A, Wolf J, Hansmann ML, Diehl V, Küppers R, Rajewsky K. Somatic mutations of the CD95 gene in Hodgkin and Reed-Sternberg cells. Cancer Res 2000;60:5640-5643. 5