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Sharid1a

Manufactured by Merck Group
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ShARID1A is a laboratory equipment product offered by Merck Group. It serves as a tool for researchers and scientists in their work processes. The core function of ShARID1A is to facilitate specific tasks within the laboratory setting, though the details of its intended use are not provided in this factual and unbiased description.

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Lab products found in correlation

Qpcr lentiviral titration kit, by Applied Biological Materials (3 mentions) Fugene hd transfection reagent, by Promega (2 mentions) Puromycin, by Merck Group (2 mentions) Lentiviral packaging mix, by Merck Group (1 mentions)

3 protocols using sharid1a

1

Lentiviral Transduction for shRNA Expression

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Lentiviral particles expressing shRNAs were produced in 293T cells according to the manufacturers’ instructions. Lenti-X™293T cells were transfected with lentiviral packaging mix composed of pNHP and pVSVG (generous gifts from Dr. Fredric Manfredsson) and MISSION pKLO.1 plasmid containing non-targeting shRNA (control) or pooled ARID1A shRNAs (shARID1A) (Sigma) using poly-ethylenimine (PEI) in DMEM + 4.5g/L D-Glucose, 110mg/L Sodium Pyruvate, 10% FBS, 1% L-glutamine. After 24 hr, media was replaced with DMEM/F12, 10% FBS, 1% L-glutamine, 1% P/S. Viral particles were collected after 48 and 96 hr, and viral titers were calculated using the qPCR Lentiviral Titration Kit (ABM).
For lentiviral transduction of 12Z cells, cells were treated with a multiplicity of infection of 100 units per cell. After 24 hours, media was replaced. For plasmid co-transfection experiments, cells were transfected the following day with 500ng pBabe vector containing PIK3CAH1047R (pPIK3CAH1047R) or pBabe empty vector using the FuGene HD transfection reagent (Promega) according to the manufacturers’ instructions at a ratio of 2:1 volume:mass, and media was replaced after 4 hr. In A-485 co-treatment studies, A-485 was included in the media 24 hr-post transfection in 0.1% DMSO. To generate stable expression cell lines, transduced cells were treated with 600 ng/mL puromycin (Sigma) for three weeks.
Wilson M.R., Reske J.J., Holladay J., Neupane S., Ngo J., Cuthrell N., Wegener M., Rhodes M., Adams M., Sheridan R., Hostetter G., Alotaibi F.T., Yong P.J., Anglesio M.S., Lessey B.A., Leach R.E., Teixeira J.M., Missmer S.A., Fazleabas A.T, & Chandler R.L. (2020). ARID1A Mutations Promote P300-Dependent Endometrial Invasion through Super-Enhancer Hyperacetylation. Cell reports, 33(6), 108366.
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2

Lentiviral Transduction of shRNAs

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Lentiviral particles expressing shRNAs were produced in 293T cells according to the manufacturers’ instructions. Briefly, Lenti-XTM 293T cells were transfected with lentiviral packaging mix (Sigma) and MISSION pKLO.1 plasmid containing non-targeting shRNA (shNONtg) or pooled ARID1A shRNAs (shARID1A) (Sigma) using polyethylenimine (PEI) in DMEM + 4.5 g/L d-Glucose, 110 mg/L Sodium Pyruvate, 10% FBS, 1% l-glutamine. After 4 h, media was replaced with DMEM/F12, 10% FBS, 1% L-glutamine, 1% P/S. Viral particles were collected after 48 and 96 h, and viral titers were calculated using the qPCR Lentiviral Titration Kit (ABM).
Wilson M.R., Reske J.J., Holladay J., Wilber G.E., Rhodes M., Koeman J., Adams M., Johnson B., Su R.W., Joshi N.R., Patterson A.L., Shen H., Leach R.E., Teixeira J.M., Fazleabas A.T, & Chandler R.L. (2019). ARID1A and PI3-kinase pathway mutations in the endometrium drive epithelial transdifferentiation and collective invasion. Nature Communications, 10, 3554.
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3

Lentiviral Transduction for shRNA Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols
Lentiviral particles expressing shRNAs were produced in 293T cells according to the manufacturers’ instructions. Lenti-X™293T cells were transfected with lentiviral packaging mix composed of pNHP and pVSVG (generous gifts from Dr. Fredric Manfredsson) and MISSION pKLO.1 plasmid containing non-targeting shRNA (control) or pooled ARID1A shRNAs (shARID1A) (Sigma) using poly-ethylenimine (PEI) in DMEM + 4.5g/L D-Glucose, 110mg/L Sodium Pyruvate, 10% FBS, 1% L-glutamine. After 24 hr, media was replaced with DMEM/F12, 10% FBS, 1% L-glutamine, 1% P/S. Viral particles were collected after 48 and 96 hr, and viral titers were calculated using the qPCR Lentiviral Titration Kit (ABM).
For lentiviral transduction of 12Z cells, cells were treated with a multiplicity of infection of 100 units per cell. After 24 hours, media was replaced. For plasmid co-transfection experiments, cells were transfected the following day with 500ng pBabe vector containing PIK3CAH1047R (pPIK3CAH1047R) or pBabe empty vector using the FuGene HD transfection reagent (Promega) according to the manufacturers’ instructions at a ratio of 2:1 volume:mass, and media was replaced after 4 hr. In A-485 co-treatment studies, A-485 was included in the media 24 hr-post transfection in 0.1% DMSO. To generate stable expression cell lines, transduced cells were treated with 600 ng/mL puromycin (Sigma) for three weeks.
Wilson M.R., Reske J.J., Holladay J., Neupane S., Ngo J., Cuthrell N., Wegener M., Rhodes M., Adams M., Sheridan R., Hostetter G., Alotaibi F.T., Yong P.J., Anglesio M.S., Lessey B.A., Leach R.E., Teixeira J.M., Missmer S.A., Fazleabas A.T, & Chandler R.L. (2020). ARID1A Mutations Promote P300-Dependent Endometrial Invasion through Super-Enhancer Hyperacetylation. Cell reports, 33(6), 108366.
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