Fig. 5
From: DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs
Derivation of TFRC cell clones. a Outline of the clone-derivation protocol used for TFRC and MALAT1 knock out cells, indicating approximate time required. b First and c second stage PCRs to genotype clones. Primer combination schemes are indicated below the electrophoresis gels. H: TFRC_B cell clone genotyped as heterozygote; WT, cell clones genotyped as wild type; +, positive control wild type cells; H2O, water. d qRTPCR for TFRC mRNA, normalised to GAPDH. Error bars indicate the standard deviation of three technical replicates. e Flow cytometry analysis of surface levels of TFRC protein. Left: histogram of cell fluorescence intensity counts. Right: Calculation of relative stain index. f Sequencing analysis of mutant junction of the heterozygous clones. In red, region complementary to the gRNA variable region; Green, PAM sequences; Blue, indel. Expected cut location is marked with vertical bar