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. 2020 Aug 3;9:e58659. doi: 10.7554/eLife.58659

Figure 3. A small-scale CRISPR loss-of-function screen for dynamically expressed and translated lncRNAs during pancreatic differentiation.

(A) qRT-PCR analysis of candidate lncRNAs during pancreatic differentiation of H1 hESCs relative to the ES stage. Data are shown as mean ± S.E.M. (mean of n = 2–6 independent differentiations per stage; from H1 hESCs). Individual data points are represented by dots. See also Figure 3—source data 2. (B) CRISPR-based lncRNA knockout (KO) strategy in H1 hESCs and subsequent phenotypic characterization. (C) Immunofluorescence staining for OCT4 and SOX17 in DE from control (ctrl) and KO cells for the indicated lncRNAs (representative images, n ≥ 3 independent differentiations; at least two KO clones were analyzed). (D) qRT-PCR analysis of DE lineage markers in DE from control and lncRNA KO (-/-) cells. TF genes in cis to the lncRNA locus are highlighted in red. Data are shown as mean ± S.E.M. (n = 3–16 replicates from independent differentiations and different KO clones). Individual data points are represented by dots. NS, p-value>0.05; t-test. See also Figure 3—source data 3. (E) Flow cytometry analysis at DE stage for SOX17 in control and KO (-/-) cells for indicated lncRNAs. The line demarks isotype control. Percentage of cells expressing SOX17 is indicated (representative experiment, n ≥ 3 independent differentiations from at least two KO clones). (F) Immunofluorescence staining for FOXA2 or GATA6 in DE from control and LINC00261, GATA6-AS1, and DIGIT KO cells. (G) Immunofluorescence staining for insulin (INS) in endocrine cell stage (EC) from control and KO hESCs for the indicated lncRNAs (representative images, n ≥ 3 independent differentiations from at least two KO clones). Boxed areas (dashed boxes) are shown in higher magnification. (H) qRT-PCR analysis of INS in EC stage cultures from control and lncRNA KO (-/-) hESCs. Data are shown as mean ± S.E.M. (n ≥ 4 replicates from independent differentiations of at least two KO clones). Individual data points are represented by dots. NS, p-value>0.05; t-test. See also Figure 3—source data 4 (I) Flow cytometry analysis at EC stage for INS in control and KO (-/-) cells for indicated lncRNAs. The line demarks isotype control. Percentage of cells expressing insulin is indicated (representative experiment, n ≥ 3 independent differentiations each from at least two KO clones). Scale bars = 100 µm. See also Figure 3—figure supplement 1 and Figure 3—source data 1–4.

Figure 3—source data 1. Differentially expressed genes after lncRNA deletion.
(A) Coordinates of CRISPR deletions. (B) Differentially expressed genes in RP11-445F12.1 knockout at definitive endoderm stage. (C) Differentially expressed genes in GATA6-AS1 knockout at definitive endoderm stage. (D) Differentially expressed genes in LINC00261 knockout at definitive endoderm stage. (E) Differentially expressed genes in LINC00617 knockout at PP2 stage. (F) Differentially expressed genes in GATA6-AS1 knockout at PP2 stage. (G) Differentially expressed genes in LINC00479 knockout at PP2 stage. (H) Differentially expressed genes in RP11-834C11.4 knockout at PP2 stage. (I) Differentially expressed genes in SOX9-AS1 knockout at PP2 stage. (J) Differentially expressed genes in MIR7-3HG knockout at PP2 stage. (K) Differentially expressed genes in LHFPL3-AS2 knockout at PP2 stage.
Figure 3—source data 2. Source data used for the qRT-PCR quantification of gene expression presented in Figure 3A.
Figure 3—source data 3. Source data used for the qRT-PCR quantification of gene expression presented in Figure 3D.
Figure 3—source data 4. Source data used for the qRT-PCR quantification of INS expression presented in Figure 3H.

Figure 3.

Figure 3—figure supplement 1. Minor gene expression changes in definitive endoderm or pancreatic progenitor cells after lncRNA deletion.

Figure 3—figure supplement 1.

(A) Genome Browser snap shots of RNA-seq signal at the indicated lncRNA loci in control (ctrl) and lncRNA knockout (KO; -/-) DE (green tracks) and PP2 (red tracks) stage cells. Genomic deletions are indicated by gray boxes. (B) Bar graphs showing expression of indicated lncRNAs in control and lncRNA KO DE (green) and PP2 (red) cells quantified by RNA-seq. Data are shown as mean RPKM (n = 2–6 independent differentiations of two independent KO clones, except for SOX9-AS1 for which one clone was differentiated twice). Individual data points are represented by dots. (C) Volcano plots displaying gene expression changes in control versus lncRNA KO DE (green) or PP2 (red) cells. Differentially expressed genes (DESeq2; >2 fold change (FC), adjusted p-value<0.01; vertical and horizontal dashed lines indicate the thresholds; n = 2 independent differentiations of two independent KO clones, except for SOX9-AS1 for which one clone was differentiated twice) are shown in green (DE) and red (PP2). TF genes in cis to deleted lncRNAs are shown in gray (gray dots represent genes with ≤ 2 fold change and/or adjusted p-value≥0.01).