title: Illumina NovaSeq 6000 paired end sequencing; Functional characterization of the role of the tyrosine-protein kinase BAZ1B in hormone- responsive breast cancer cells
design: Functional characterization of the role of the tyrosine-protein kinase BAZ1B in hormone- responsive breast cancer cells
library_name: Sample 9_p
library_construction_protocol: Human Breast cell lines ERa-positive MCF-7 and tamoxifen resistant MCF-7 tam 1 (ATCC HTB-22; ATCC-CRL-3435) were purchased from the American Type Culture Collection (ATCC) while ICI-resistant MCF-7/182R-6 cells (16022506) were purchased from European Collection of Authenticated cell cultures. All cell lines were cultured according to manufacture protocol. Tamoxifen and Fulvestrant resistant cells medium was supplemented, respectively, with 1 M 4-hydroxytamoxifen (4-OH TAM) or 100 nM fulvestrant (ICI 182,780) (Sigma-Aldrich). MCF-7 wt stably transfected by ERE-TK-LUC plasmid (ref) containing the luciferase reporter gene under the control of the estrogen-responsive element was cultured in in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% FBS (HyClone, Milan, Italy) and 100 U/ml penicillin, 100 mg/mL streptomycin, and 250 ng/mL Amfotericin-B. Functional assays and RNA sequencing experiment were performed after BAZ1B knock-down. MCF-7 cells were seeded into 96-well plates (1,4  103 cells per well) in standard growth medium and transiently transfected by using Lipofectamine RNAiMax (13778-150, Invitrogen) diluted in Optimem medium, according to the manufacturer's instructions. 0.5 pM/well of two validated BAZ1B siRNAs (s17208, s17210) or Silencer Select Negative Control (SiSel_NC1) (Ambion, Thermo Fisher Scientific) were added in sixfold each one and incubated for 72h at 37 C/5% CO2.  Total RNA was extracted using TRIzol (Thermofisher Scientific, Waltham, MA, USA), according to the manufacturers instructions. Furthermore, 0.31 mL of TRIzol Reagent per 1,4 X 103 cells was added directly to the culture dish to lyse the cells by pipetting several times to homogenize. Before use, RNA concentration was determined by using Quant-IT RNA Assay Kit-High Sensitivity and a Qubit Fluorometer (Life Technologies, Monza, Italy) and its quality and integrity assessed with the Agilent 4200 Tapestation System (Agilent Technologies, Milan, Italy). For RNA sequencing, cell lines silencing with siRNA BAZ1B 1 and 2 or treated with 10-6 M ICI 182,7800 were prepared in sixfold and pooled. Negative control (scramble, nontargeting) was used as control.   Indexing libraries were prepare starting to 150 ng of total RNA according to Illumina Stranded Total RNA prep Ligation with Ribo-Zero Plus kit (Illumina Inc., San Diego, CA, USA).