Fig. 1

Pooled shRNA screen identifies several genes involved in breast cancer tumourigenesis. A. Establishment of the 3D on-top assay in 2% serum with growth factor reduced Matrigel. This method facilitates preferential acini outgrowth of MDA-MB-231, but not T-47D breast cancer cells. B. Schematic of workflow showing shRNA screening in T-47D cells. T-47D cells were transduced at an MOI of < 0.5, selected in puromycin for 3 days. For the Polarity and Kinome libraries, 1.5 × 10⁶ selected T-47D cells were plated on a T75 flask of Growth Factor Reduced (GFR) matrigel overlaid with DMEM 2% FCS. Day zero samples were frozen and Day 14 acini were collected by dissolving the matrigel in ice cold versene and pelleting the cells. Genomic DNA was prepared from day 0 and day 14 samples for amplification, sequencing and quantification of shRNA sequences. The abundance of hairpins before and after acini culture was compared to detect enrichment