Transdux reagent

The pre-miR224 expression construct, miRZip-224 anti-miR-224 construct and control vector were packaged with pPACKH1 Lentivector Packaging Plasmid mix (System Biosciences) in a 293T packaging cell line. The Transdux reagent (System Bioscience) was used for virus transduction, and infected cells were selected by fluorescence-activated cell sorting (FACS) analysis (FACSCalibular, BD Bioscience). Transfection of shRNA against CASP7 was carried out with Lipofectamine LTX according to the manufacturer's instruction (Invitrogen) and transfected cells were selected by puromycin. The siRNA against RELA was transfected to cells by using Lipofectamine RNAiMAX according to the manufacturer's instruction (Invitrogen).

Packed lentiviral particles, containing enhanced green fluorescent protein (eGFP) driven by distinct promoters (mPol2, Grp78, FerH, CAG, CMV13, PGK, EF1α, TRE-Tight), were ordered from Leidos Biomedical Research Inc. (Frederick, MD, USA). 6-day-old primary cortical neuron cultures from C57BL/6 mice were infected at MOI 10, 20 and 40 with the TransDux reagent. eGFP expression was evaluated after 5 days of infection with a fluorescence microscope (Zeiss, San Diego, CA, USA). Packed lentiviral particles containing EF1α-SV40T were ordered from Leidos Biomedical Research Inc and 6-day-old primary cortical neuron cultures from gba^−/− and gba^+/+ mice were infected at MOI 40 with TransDux reagent (System Biosciences). After 4 days of infection, the cultures were treated with 1 µg/ml of puromycin (Sigma Aldrich) for 4 weeks; the media was changed every 3 days.

To infect mammosphere cultures with lentivirus, primary mammospheres were disassociated as for secondary mammosphere assays. Before plating, single cell suspensions in DMEM/F12 media were mixed with lentiviral particles in PBS at a multiplicity of infection of greater than 5∶1, plus a 1∶200 dilution of Transdux reagent (System Biosciences) for 30 minutes at 37°C. After infection, the cell suspension was plated as for secondary sphere assays. The 7TGC lentiviral reporter vector was obtained from Addgene (Plasmid#24304) [49] (link). Lentiviral vectors for knockdown of Ror2 mRNA were obtained from Dr. T. Stappenbeck [50] (link). For clarity we renamed the knockdown vector shRor2#7 [50] (link) as shRor2 and the control vector SCH002-EGFP as shControl.

Negative screening was performed using the Decode RNAi Pooled Lentiviral shRNA Screening Libraries: Annotated Genome Negative Selection Kit (Thermo Scientific). Briefly, T-REx HeLa cells (Invitrogen) were infected with a lentiviral siRNA expression library (Thermo Scientific) using TransDux reagent (System Biosciences). Green fluorescent protein-positive cells were selected by puromycin treatment and divided into two populations, one of which was irradiated with 0.75 Gy using a ¹³⁷Cs irradiator (Gy/min, Best Theratronics); the other served as a non-irradiated control. Four days after irradiation, genomic DNA was purified from each population using a DNA purification kit (Dojindo). Barcode sequences were amplified from 0.5 μg of genomic DNA, purified by gel extraction, and labeled using a Genomic DNA Enzymatic Labeling Kit (Agilent Technologies). After purification using Amicon Ultra-0.5 ml centrifugal filters (Millipore), the labeled barcode sequences were hybridized with microarray slides for 17 h and the slides were then washed according to the Agilent CpG microarray protocol. Cluster analysis of the extracted genes was performed using the information in the GeneCards database (http://www.genecards.org/) and KEGG (http://www.genome.jp/kegg/).