Lentiviral Transduction for EGFR perturbation

Lentiviral Transduction for EGFR perturbation Wednesday, October 28, 2021 Wiley/Hess Lab, Pacific Northwest National Laboratory Document Last reviewed:

Protocol revised from: 20201106_Protocol for Viral Transduction_SP Final Project WP: NH8697 Description Lentiviral constructs have been prepared by VectorBuilder. Will be transducing 2 cell lines (MCF10A_dCas9-KRAB and MCF10A_dCas9-VPR) with sgRNA libraries to obtain variable EGFR expression. Cell Lines

  1. MCF10A dCAS9-KRAB a. Antibiotic selection: 10µL Blasticidin – 100mg/mL concentration + Puromycin b. KRAB: 10 µg/mL Blasticidin c. Library: 2 µg/mL Puromycin – 10mg/ml stock diluted 1:5000 dilution

  2. MCF10A dCAS9-VPR a. Antibiotic selection: 100µL of 50mg/mL G418 + Puromycin b. VPR: 500 µg/mL G418 c. Library: 2 µg/mL Puromycin – 10mg/ml stock diluted 1:5000 dilution

Lentivirus a. lentiGuide-Puro-sgRNA-Lib-EGFRi b. >108 TU/ml c. Made from library: Lib20210329-TL001

		a.	lentiGuide-Puro-sgRNA-Lib-EGFRa
		b.	>108 TU/ml
		c.	Made from library: Lib20210329-TL002

Media Note: Engineered base media will not have PenStrep, but will have different conentrations of EGF depending on the lentivirus  MCF10A media for EGFRi library construction: DMEM/F12 (Invitrogen #11330-032), 5% Horse Serum (Invitrogen #16050-122), Hydrocortisone (0.5mg/ml Sigma #H-0888), Cholera Toxin (100ng/ml, Sigma #C-8052), Insulin (10ug/ml, Sigma #I-5016-5ML), 1% Pen/Strep

SEEK ID: https://emsl-seek.pnnl.gov/sops/1?version=2

Filename: MCF10A Transduction for Adaptors-final.docx 

Format: Word document

Size: 1.43 MB

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Created: 29th Oct 2021 at 00:16

Last updated: 20th Oct 2022 at 03:49

Last used: 15th Apr 2024 at 11:17

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Version History

Version 2 (latest) Created 20th Oct 2022 at 03:49 by Steven Wiley

This version is designed for growth selection using EGF concentration as a variable rather than flow cytometry. It also includes the protocols for amplify the sgRNAs using nested PCR.

Version 1 (earliest) Created 29th Oct 2021 at 00:16 by Steven Wiley

This protocol is designed for flow sorting alone.

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