Pcr8 topo vector

Genomic DNA isolation was performed using the Gentra PureGene kit (Qiagen). DNA was then PCR amplified with Platinum Taq
DNA Polymerase High Fidelity (Invitrogen) following manufacturer’s instructions. PCR products were cloned into the pCR8
TOPO vector (Invitrogen) and subjected to Sanger sequencing on a 3730XL DNA Sequencer (Applied Biosystems) at the University of
Michigan DNA Sequencing Core. Sequence alignment was performed using the DNASTAR Lasergene software suite.

Lentiviruses were packaged and used to infect target cells as previously described (26 (link)). PLK0.1 vectors containing shRNA’s targeting ZEB1 (TRC Version: TRCN0000017565; Clone Name: NM_030751.2-572s1c1 and TRC Version: TRCN0000017566; Clone Name: NM_030751.2-70s1c1) were purchased from Sigma Aldrich. PLK0.1 vector containing shRNA-targeting GFP has been previously described (26 (link)). The SORE6 cancer stem cell reporter construct was a kind gift from Dr. Lalage Wakefield (27 (link)). Lentiviral pLenti-CMV-GFP was obtained from Addgene; Plasmid #17447. pBABE-puro-hTERT was purchased from addgene (plasmid #1771). pLenti-Neo-VEC was generated using gateway cloning and recombining the multiple cloning site of PCR8 topo vector (Invitrogen; Cat#46-0899) into lentiviral vector pLenti-CMV-Dest (Addgene; Plasmid#17451). pLenti-Neo-OSM as generated by sub-cloning OSM cDNA (OriGene; Cat# SC-121421) into gateway entry vector pLenti-ENTR4 (Addgene; Plasmid# 17424) and recombined into lentiviral destination vector (Addgene; Plasmid#17451). pFLUG-GFP-LUC (Firefly) fusion construct was a kind gift from Dr. Huiping Liu (28 (link)).

Genomic DNA was used to verify somatic alterations including single nucleotide variations. 10ng of total DNA was used for PCR amplified with Platinum Taq DNA Polymerase High Fidelity (Invitrogen) according to manufacturer’s instructions. Primer sequences for each target are listed in Supplementary Table 3. For amplicons greater than 200bp in length, PCR products were PCR purified using the Qiagen PCR purification kit and submit for Sanger sequencing at the University’s DNA sequencing core on the 3730XL DNA Sequencer (Applied Biosystems). For amplicons less than 200bp in length, PCR products were cloned out using pCR8 TOPO vector (Invitrogen) and submitted for Sanger sequencing using forward and reverse primers from the cloning vector. Sequences were aligned using the DNASTAR Lasergene software suite against the RefSEQ annotation from HG19.