lentiCRISPR v2

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Protocol tips
There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

Glucose and Galactose Cell Death Screening
K562 cells expressing Cas9 and EGFP were infected with the genome-wide Avana-4 CRISPR lentiviral library (Doench et al., 2016). Two infection replicates were performed. Infected cells were selected for 4 days with 2 μg/mL puromycin (Life Technologies), after which a sample of cells was collected for determining the initial sgRNA abundance. The cells were expanded for an additional 6 days, washed with PBS, and cultured in DMEM with 10 mM glucose or galactose, 10% dialyzed FBS (dFBS, Life Technologies), 1 mM sodium pyruvate, 50 μg/mL uridine, and 100 U/mL penicillin/streptomycin. Pre-swap cells were harvested prior to the medium swap. After 24-hr culture in glucose or galactose medium, Annexin V+ dead cells were purified using magnetic Annexin V microbeads and mass spectrometry (MS)-positive selection columns (Miltenyi Biotec). Genomic DNA was isolated from all samples, and the sgRNA sequences were amplified by PCR and sequenced on a HiSeq 2500 (Illumina). MAGeCK (v0.4.4) was used to score genes under each condition relative to the pre-swap reference samples (Li et al., 2014). See the Supplemental Experimental Procedures for details.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Deletion NGRN using lentiCRISPR v2 from Addgene.

Manufacturer protocol

Download the product protocol from Addgene for lentiCRISPR v2 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Human - Deletion NGRN using lentiCRISPR v2 from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.