Illumina HiSeq 2500 sequencing; Estrogen receptor β role in tumour suppression of triple negative breast cancer - ChIPseq experiment
Estrogen receptor β role in tumour suppression of triple negative breast cancer - ChIPseq experiment
MDA-MB-468 (HTB-132) and cell lines were purchased from the American Type Culture Collection (ATCC). Each cell line was grown in the appropriate culture media, according to manufacturer's protocol, and kept in an incubator at 37 °C, in the presence of 5% CO2. Additional details are described in Supplementary Materials. Periodically, cells were tested for mycoplasma contamination using the PCR Mycoplasma detection kit (Applied Biological Materials). The selected cell lines were used to generate full-length ERβ-expressing experimental model, using the Clontech Lenti-X Tet-On Advanced Inducible Expression System. First, the 3xFlag-ERβ1 DNA fragment was cloned  into a pLVX-Tight-Puro vector, downstream to a tetracycline inducible promoter. The cloning procedure was performed using the Clontech In-Fusion HD Cloning Kit. Subsequently, to produce lentiviral particles expressing both rTA and Erβ1 genes,Clontech's Lenti-X Packaging Single Shots (VSV-G) were used according to manufacturer's protocol. Finally, the selected TNBC cell lines were infected first with the rtTA lentiviral particles, encoding for a a transcription factor that binds to a tightly regulated inducible promoter, after were transduced with 3xFlag-ERβ lentiviral particles and tested for the protein expression upon doxycycline (Sigma-Aldrich) induction None None Chromatin was prepared and isolated as described by Tarallo et al. [Tarallo R, Giurato G, Bruno G, Ravo M, Rizzo F, Salvati A, Ricciardi L, Marchese G, Cordella A, Rocco T, Gigantino V, Pierri B, Cimmino G, Milanesi L, Ambrosino C, Nyman TA, Nassa G, Weisz A. The nuclear receptor ERβ engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading. Genome Biol. 2017 Oct 6;18(1):189. doi: 10.1186/s13059-017-1321-0. PubMed PMID: 29017520; PubMed Central PMCID: PMC5634881.] starting from a total of approximately 24 x 106 MDA-MB-468 ERβ clone cells previously treated or not with doxycycline (2.0 µg/mL) for 9 days. Before the immunoprecipitation, for each condition two aliquots of the diluted nuclear extract were set aside for DNA and protein input analyses. To immunoprecipitate 3xFLAG-ERβ, treated and not treated chromatin samples were incubated overnight at 4°C on rotating wheel with respectively 60 and 65 µl of anti-mouse Magnetic Beads (Invitrogen) previously armed, according to Schmidt et al. [Schmidt D, Wilson MD, Spyrou C, Brown GD, Hadfield J, Odom DT. ChIP-seq: using high-throughput sequencing to discover protein-DNA interactions. Methods. 2009 Jul;48(3):240-8. doi: 10.1016/j.ymeth.2009.03.001. Epub 2009 Mar 9. PubMed PMID: 19275939; PubMed Central PMCID: PMC4052679.], with 6 and 6.5 µg of anti-FLAG (F3165, Sigma-Aldrich). Three independent biological replicates were performed for each condition. Beads washing, DNA elution and extraction were performed as described by Ambrosino et al. [Ambrosino C, Tarallo R, Bamundo A, Cuomo D, Franci G, Nassa G, Paris O, Ravo M, Giovane A, Zambrano N, Lepikhova T, Jänne OA, Baumann M, Nyman TA, Cicatiello L, Weisz A. Identification of a hormone-regulated dynamic nuclear actin network associated with estrogen receptor alpha in human breast cancer cell nuclei. Mol Cell Proteomics. 2010 Jun;9(6):1352-67. doi: 10.1074/mcp.M900519-MCP200. Epub 2010 Mar 22. PubMed PMID: 20308691; PubMed Central PMCID: PMC2877992.] Before DNA elution, an aliquot of beads of both conditions was taken for proteins elution, performed adding sample buffer (0.5 M Tris-HCl pH 6.8, 30% glycerol, 12% SDS, 9,3% DTT and 0.012% Bromophenol blue) and boiling at 100°C for 10 minu The size distribution and concentration of each ChIP-DNA sample were determinated as described early [Tarallo et al], then 2 µg of each sample were processed with TruSeq ChIP Sample Prep Kit (Illumina Inc.) for indexed libraries preparation.
Experimental Factor: phenotype
not expressing FLAG-ERB
Experimental Factor: immunoprecipitate
anti-FLAG (Sigma-Aldrich)