design: Essential gene screening identifies the Bromodomain-Containing Protein BRPF1 as a new therapeutic target for endocrine therapy-resistant breast cancer - organoids
library_construction_protocol: Tumor organoids isolated from human specimens were used to examine the treatment response in breast cancer. Breast tissues were obtained from surgical procedures at Clinica Mediterranea Cardiocentro and were processed on the day of surgery.tumor samples were cut in small pieces and then digested in 10 mL of media (Advanced DMEM/F12, 1 Glutamax, 10 mM HEPES, and 1% antibiotics/antimycotic) containing 2 mg/mL of collagenase IV (Sigma, C9407) for 3 hours at 37 °C, in an orbital shaker.  The digested suspension was then mechanically shared using pipette tips, washed, and centrifuged at 400g. The final pellet of organoids was suspended in Cultrex growth factor reduced BME type 2 (Trevigen, Cat. No. 3533-010-02) and 40 ml were placed in the centre of a 24-well culture plate (Grainer, cat. No. M9312). Finally, after 20 min at 37 °C to allow BME solidification, 400 ml of BC organoids medium was added to each droplet. Medium was changed every 3-4 days and split using Tryple Express (Invitrogen, 12648-010). BME drops containing organoids were washed two times with cold PBS, gently detached from the bottom of the well and collected in Eppendorf. BME was removed through mechanically disaggregation using p1000 pipette tips and incubating the plate at 4 °C for 30 min. The released organoids were centrifuged and fixed in 4% paraformaldehyde. Then, organoids were dehydrated, embedded in paraffin, sectioned and HE stained. Immunohistochemistry was performed using an antibody against ER and Ventana platform (Roche). For drug treatment, organoids were disaggregated in single cells using Tryple Express, resuspended in growth media containing 5% of BME and plated in triplicate in a 96 well previously pre-coated with 40 ml of BME. After 3-day, organoids were treated with an increasing concentration of drug GSK6853 (10 µM, 20 µM and 30 µM) and ICI (100 nM) for 10 days. Viability assay was measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Cat. No. G3581). The apoptosis response was detected by measuring caspase-3/7 activity by Caspase-Glo®3/7 assay (Promega Cat. No G80092). For RNA analysis, organoids were plated in 40 ml of BME in a 24-well culture plate in duplicate. After 3 days, organoids were treated with GSK6853 drug (30 mM) and Vehicle (DMSO) as control. After 10 days of treatment, organoids were collected in TRIZOL and RNA extraction was performed as described above. Total RNA was extractedusing TRIzol reagent (Life Technologies, Carlsbad, CA), according to the manufacturer's instructions. In brief, 50 µl of TRIzol™ Reagent for 3x104 cells was added directly to the culture dish to lyse the cells by pipetting several times, both for RT-qPCR and RNA sequencing. After phase separation step, the aqueous supernatant was collected and purified using RNA Clean & Concentrator − 5 kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions. Before use, RNA purity was assessed by NanoDrop™ 2000/2000c spectrophotometer (Thermo Fisher Scientific), its concentration was measured using Quant-IT RNA Assay Kit-High Sensitivity and a Qubit Fluorometer (Life Technologies, Monza, Italy) and integrity assessed with the Agilent 4200 Tapestation System (Agilent Technologies, Milan, Italy).   indexed libraries were prepared starting from 1 total RNA according to Illumina Stranded Total RNA prep Ligation with Ribo-Zero Plus kit (Illumina Inc., San Diego, CA, USA).