Quantitative and non-quantitative RT-PCR. cdna was generated from 500ng RNA (iscript;

Transcription

1 Supplemental Methods Quantitative and non-quantitative RT-PCR. cdna was generated from 500ng RNA (iscript; Bio-Rad, Hercules, CA, USA) and standard RT-PCR experiments were carried out using the 2X GoTaq Green Master Mix (Promega). Quantitative RT-PCR experiments were conducted using Taqman gene expression assays (Life Technologies) with the following probes: HOXD1 (Hs _s1), HOXD3 (Hs _m1), HOXD4 (Hs _g1), HOXD13 (Hs _m1). Primer sequences for non-quantitative RT-PCR are listed below. RT-PCR Primer Sequences Gene Forward primer sequence, 5 to 3 Reverse primer sequence, 5 to 3 HOXA1 TCCTGGAATACCCCATACTTAGC GCACGACTGGAAAGTTGTAATCC HOXA2 GCACGACTGGAAAGTTGTAATCC ACCCCGAAGGGTGGAGATT HOXB5 ACCCCGAAGGGTGGAGATT CGGAGTCCTCAAGGCTTTTACAT HOXB6 CGGAGTCCTCAAGGCTTTTACAT AAC TCC TCG GGG CGT TAT HOXB9 AACTCCTCGGGGCGTTAT CATCCCATTGTAATTGTAGCCGT HOXB13 CATCCCATTGTAATTGTAGCCGT TCCTATTTCGTGAACTCCACCT HOXC9 TCCTATTTCGTGAACTCCACCT GCGGGGTAATGTCTCAGCG HOXC10 GCGGGGTAATGTCTCAGCG CCATTTCTGGGACGCTTAGCA HOXC11 CCATTTCTGGGACGCTTAGCA TGTAAGGGTGGTAGACGGACG HOXC13 TGTAAGGGTGGTAGACGGACG CCAGTTACCTGGACGTGTCTG HOXD9 CCAGTTACCTGGACGTGTCTG GGACCTGGTGGGTTCTGTTC HOXD10 GGACCTGGTGGGTTCTGTTC ACTCGCTCATCTCTCACGACA HOXD11 ACTCGCTCATCTCTCACGACA GACGGAAAATCGCTACAGTCC HOXD13 GACGGAAAATCGCTACAGTCC CGCCGAACATCTGGAATCG 1

2 GAPDH TGCACCACCAACTCGTTAGC GGCATGGACTGTGGTCATGAG 18S GCAATTATTCCCCATGAACGA GGCCTCACTAAACCATCCAAT ChIP-PCR Histone Modifications Cells were fixed on the dish at room temperature for 10 min in growth medium containing 1% formaldehyde. To quench the fixation, 2M glycine was added to a final concentration of 0.18M and allowed 5 min incubation at room temperature. Plates were rinsed 3x with cold PBS and then scraped on ice into PBS containing protease and phosphatase inhibitors (Roche). Fixed cells were pelleted via centrifugation at 4 C, 4000 x g, for 10 min. Pellets were then resuspended in 150 µl SDS lysis buffer (50mM tris-hcl ph 8.1, 10mM EDTA ph 8, and 10% SDS) containing protease and phosphatase inhibitors and incubated on ice min with frequent vortexing. Lysates were sonicated on wet ice using a Qsonica cup horn sonicator, 100% amplitude, second pulses with 1 min rest in between. DNA fragments were in the bp range. Insoluble components were pelleted via centrifugation, 14,200 x g, 4 C for 10 min. Supernatant was transferred to fresh microcentrifuge tubes and diluted 10-fold in dilution buffer (16.7 mm tris-hcl ph 8.1, 167 mm NaCL, 1.2 mm EDTA ph 8, 1.1% triton). Samples were then pre-cleared for 2 hrs, rotating at 4 C using 30µL of protein G dynabead slurry (Invitrogen). Prior to the pre-clearing step, beads were blocked in dilution buffer containing 5 mg/ml BSA + 10µg/mL glycogen for 2 hrs at 4 C, washed and resuspended in fresh dilution buffer. Pre-cleared samples were then transferred to fresh tubes and 1% of each sample (15µL) was frozen at -20 C for later analysis of input. The remaining sample was incubated overnight, rotating at 4 C with 2 µg antibody or control IgG. The next day, antibody-dna complexes were captured using 50 µl of protein G dynabead slurry, rotating at 4 C for 2 hrs. Beads were then washed once with each of the 2

3 following buffers in order for 5 4 C: Low Salt wash (150 mm NaCl, 0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris (ph 8.0)), High Salt wash (500 mm NaCl, 0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris (ph 8.0)), LiCl wash (250 mm LiCl, 1% Deoxycholate, 1% NP-40, 1 mm EDTA, 10 mm Tris (ph 8.0)), TE wash (10 mm Tris (ph 8.0), 1 mm EDTA). Antibody/DNA complexes were eluted by adding freshly made 100µL elution buffer (1%SDS, 0.1M NaHCO 3 ) to the washed beads, followed by vortexing at a medium setting at room temp, 15 min. Supernatant was then transferred to a new 1.5 ml tube and the process repeated (200µL final volume). Elution buffer (185µL) was added to the15µl 1% Input samples. 8µL 5M NaCl and 4µL RNaseA (10mg/mL) were then added to all samples, followed by crosslink reversal 5-6 hrs at 65 C. To digest protein, 4µL 0.5M EDTA, 8µL 1M Tris-HCl (ph 6.5), 4µL Proteinase K (10mg/mL) were added to each sample and incubated 45 C. DNA was recovered using the QIAquick PCR Purification Kit (Qiagen), with a final elution volume of 50µL. Target sequences were amplified via qpcr using itaq SYBR green (Bio-Rad) with 2µL DNA per reaction. Assays were performed in triplicate using the LightCycler 480 System (Roche). Data were analyzed using the percent input method according to the following equation: Percent input = 100*2^(Average Ct Input Average Ct IP). ChIP-PCR FLI1 ChIP was performed using the MAGnify TM Chromatin Immunoprecipitation System (Invitrogen, Groningen, The Netherlands) according to the manufacturer s instructions with minor modifications. Cells were crosslinked with 1% formaldehyde at room temperature for 13 minutes; the reaction was stopped with 125mM Glycine for 7min. The cells were then sheared with a Bioruptor UCD200 5 times 7 minutes of alternating 30sec sonication and 30 sec break to achieve an average shearing size of 600bp. Incubation times for antibody coupling and binding chromatin to the beads was doubled and washing steps after chromatin 3

4 incubation with the Ab coupled beads were extended to 20min each. An additional washing step between IP Buffer1 and 2 was applied using the following Buffer: 0.1% SDS, 1% Triton X- 100, 2mM EDTA, 20mMTris HCl, 500mM NaCl, ph8,1. Antibodies and primers used for ChIP-PCR studies are detailed below. Primer sequences for HOXD11.12PRE (1) and for ATAD2 (2), were taken from the published literature. ChIP-qPCR antibodies Antibody Supplier/catalog number Quantity for ChIP Anti-Trimethyl Histone Millipore µg H3(Lys27) H3K4me3 Rabbit anti- Human Anti-RNA Polymerase II clone CTD4H8 Invitrogen µg Millipore µg FLI1 MyBioSource MBS µl ChIP PCR primers Gene Forward primer sequence (5 to 3 ) Reverse primer sequence, (5 to 3 ) HOXD11 TTGGCGAGCGTTGATATAGA CTTGGGCCAGGATCAACTAA HOXD13 CAAGGGGCAGGAGAGGGG GAAGTTCAGGTTGGGAGGGG Supplemental Methods References 4

5 1. Woo CJ, Kharchenko PV, Daheron L, Park PJ, Kingston RE. A region of the human HOXD cluster that confers polycomb- group responsiveness. Cell Jan 8;140(1): PubMed PMID: Pubmed Central PMCID: Epub 2010/01/21. eng. 2. Bilke S, Schwentner R, Yang F, Kauer M, Jug G, Walker RL, et al. Oncogenic ETS fusions deregulate E2F3 target genes in Ewing sarcoma and prostate cancer. Genome Res Nov;23(11): PubMed PMID: Pubmed Central PMCID: Epub 2013/08/14. eng. 5