MCM4 shRNA (h) Lentiviral Particles

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	Cervical cancer cell lines, viz. SiHa, used for this study were kind gift from Dr. VVS Murty, Columbia University, New York, USA, and grown in recommended media supplemented with 10 % fetal bovine serum and 1 % antibiotic cocktail. These cell lines had been extensively characterized [13, 14]. To confirm the HPV status of these cell lines, we performed HPV genotyping using typespecific primers (Table 1). MCM4 short hairpin RNA (shRNA) lentiviral particles werepurchased from Santa Cruz Biotechnology. This is a pool of transduction ready viral particles containing three targetspecific constructs that encode 19–25 nucleotide (plus hairpin) shRNA designed to knock down MCM4 gene expression. Lentiviral particles containing shRNA construct encoding a scrambled sequence (Santa Cruz Biotechnology,USA) were used as a negative control that will not lead to the specific degradation of any known cellular mRNA. Cells were transfected with shMCM4 and scrambled lentiviral particles in medium supplemented with 5 μg/ml polybrene, according to supplier’s protocol. copGFP lentiviral particles (Santa Cruz Biotechnology) were used as a positive control to determine the optimal delivery condition. The amount of lentiviral particles used for the cells was optimized after trying several concentrations. Stable clones expressing shRNA were selected by puromycin dihydrochloride selection. shRNA lentiviral particles are compatible to maintain MCM4 downregulation for the subsequent generations (data not shown). Same generations of cells were used for a subset of experiments.

Protocol tips

Cervical cancer cell lines, viz. SiHa, used for this study were kind gift from Dr. VVS Murty, Columbia University, New York, USA, and grown in recommended media supplemented with 10 % fetal bovine serum and 1 % antibiotic cocktail. These cell lines had been extensively characterized [13, 14]. To confirm the HPV status of these cell lines, we performed HPV genotyping using typespecific primers (Table 1). MCM4 short hairpin RNA (shRNA) lentiviral particles werepurchased from Santa Cruz Biotechnology. This is a pool of transduction ready viral particles containing three targetspecific constructs that encode 19–25 nucleotide (plus hairpin) shRNA designed to knock down MCM4 gene expression. Lentiviral particles containing shRNA construct encoding a scrambled sequence (Santa Cruz Biotechnology,USA) were used as a negative control that will not lead to the specific degradation of any known cellular mRNA. Cells were transfected with shMCM4 and scrambled lentiviral particles in medium supplemented with 5 μg/ml polybrene, according to supplier’s protocol. copGFP lentiviral particles (Santa Cruz Biotechnology) were used as a positive control to determine the optimal delivery condition. The amount of lentiviral particles used for the cells was optimized after trying several concentrations. Stable clones expressing shRNA were selected by puromycin dihydrochloride selection. shRNA lentiviral particles are compatible to maintain MCM4 downregulation for the subsequent generations (data not shown). Same generations of cells were used for a subset of experiments.

Publication protocol

Cervical cancer cell lines, viz. SiHa, used for this study were kind gift from Dr. VVS Murty, Columbia University, New York, USA, and grown in recommended media supplemented with 10 % fetal bovine serum and 1 % antibiotic cocktail. These cell lines had been extensively characterized [13, 14]. To confirm the HPV status of these cell lines, we performed HPV genotyping using typespecific primers (Table 1). MCM4 short hairpin RNA (shRNA) lentiviral particles werepurchased from Santa Cruz Biotechnology. This is a pool of transduction ready viral particles containing three targetspecific constructs that encode 19–25 nucleotide (plus hairpin) shRNA designed to knock down MCM4 gene expression. Lentiviral particles containing shRNA construct encoding a scrambled sequence (Santa Cruz Biotechnology,USA) were used as a negative control that will not lead to the specific degradation of any known cellular mRNA. Cells were transfected with shMCM4 and scrambled lentiviral particles in medium supplemented with 5 μg/ml polybrene, according to supplier’s protocol. copGFP lentiviral particles (Santa Cruz Biotechnology) were used as a positive control to determine the optimal delivery condition. The amount of lentiviral particles used for the cells was optimized after trying several concentrations. Stable clones expressing shRNA were selected by puromycin dihydrochloride selection. shRNA lentiviral particles are compatible to maintain MCM4 downregulation for the subsequent generations (data not shown). Same generations of cells were used for a subset of experiments.

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Download the product protocol from Santa Cruz Biotechnology for MCM4 shRNA (h) Lentiviral Particles below.

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