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Fig. 3 | BMC Genomics

Fig. 3

From: Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

Fig. 3

CRISPR-Cas9 screening in BLaER cells. A Workflow of the CRISPR screening experiment. The pDECKO plasmid library was transfected into HeK293T cells to obtain a library of lentivirus. BLaER1-Cas9 cells were infected at a low multiplicity of infection and double selected with antibiotics (Blasticidin and Puromycin) for 20 days. The infected cells were induced for transdifferentiation into macrophages for 3 days (T3) and 6 days (T6). Cells were labeled with antibodies against cell surface markers: CD19 (for B-lymphocytes) and Mac1 (for macrophages). Transdifferentiation status was assessed by flow cytometry. Transdifferentiated and delayed populations were isolated by Fluorescence-Activated Cell Sorting (FACS). B Flow cytometry analysis of BLaER1-Cas9 cells infected with the pDECKO_non-targeting control (left panels) and with the pDECKO_CRISPR-library (right panels) at T0, T3 and T6 of transdifferentiation. CD19 antibody, conjugated with BV510 fluorophore, was used to identify B-cells and Mac1 antibody, conjugated with PE-Cy7 fluorophore, was used to identify macrophages. Quadrants are as follows: Q1 (macrophage-like cells with presence of Mac1 and absence of CD19 surface markers); Q2 (transition cells with the presence of Mac1 and CD19); Q3 (background and not stained cells, negative for Mac1 and CD19); Q4 (lymphocyte B-like cells with the presence of CD19 and absence of Mac1 surface markers). The percentage of cells in each of the 4 quadrants is shown. The fraction of sorted cells showing a delay of transdifferentiation (“delayed” fraction) is marked in blue (gate P4), and sorted cells that differentiate at a normal pace (“differentiated” fraction) are marked in orange (gate P5). See also Supplementary Fig. S5. C Workflow for processing the sorted cell populations for deep sequencing. Genomic DNA of sorted cells was extracted and PCR amplified in two steps. For the first PCR, specific staggered primers were used to amplify the integrated fragment which contains the pgRNAs. For the second PCR, Illumina barcoded primers were used to pool different samples (see also Supplementary Fig. S4). Samples were sequenced by 150 bp paired-end Illumina sequencing. DDE (differentiation delayed effect) was calculated as the ratio of pgRNA counts in the delayed population versus the counts in the transdifferentiated population

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