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Fig. 4 | BMC Genomics

Fig. 4

From: DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs

Fig. 4

Sequence deletion in bulk cells. a Outline of the genomic PCR (gPCR) primer strategy used for genotyping. b Deletion of TFRC promoter (construct B in Fig. 2), as validated by electrophoresis of gPCR products. Wild type gDNA and water templates are used as positive and negative controls, respectively. Green and red arrows indicate the size of PCR products expected from wild type (WT) and deleted alleles. Note that in this and subsequent panels, separated lanes originate from the same original agarose gel, rearranged for clarity. c-e gPCR on bulk cells transfected with the indicated DECKO plasmids targeting (c) TFRC promoter, (d) MALAT1 upstream promoter, (e) MALAT1 major promoter. (f-h) qRTPCR on cell samples shown in (c-e). Control indicates RNA from cells transfected with a DECKO targeting GFP. Levels were normalised to GAPDH. Error bars show the standard deviation of three technical replicates. i Expression of TFRC protein on cell surface, as determined by flow cytometry analysis of antibody-stained cells. Left: histogram of cell fluorescence intensity counts. Right: Calculation of relative stain index, a normalised measure of fluorescence intensity

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