Fig 2: Permissiveness of human islet cells to endemic coronavirus and LCMV infection(A) Left: contour plots are gated on live a, ß, and “other” cell subsets from 96-h mock- or HCoV-OC43-infected islet cell cultures. Right: summary of HCoV-OC43 NP expression in live and dead islet cell subsets (n = 4 donors).(B) GCG and INS expression levels across mock-infected, NP-, and NP+ a or ß cells.(C) MC analyses were conducted with 96-h mock- or HCoV-OC43-infected islet cells. HCoV-OC43 NP staining and tSNE visualization of live and dead mock-infected (left) and SARS-CoV-2-infected (right) non-hematopoietic cells; overlaid red dots (corresponding to the red demarcated regions in the respective left contour plots) indicate background HCoV-OC43 NP staining for mock cultures and HCoV-OC43 NP detection for infected cultures.(D and E) 96-h mock or HCoV-NL63 infection of islets; data are displayed as in (A) and (B) (n = 4 donors).(F and G) 72-h mock or LCMV infection of islets; contour plots gated on live cells show the viral burden in a, ß, and “other” cells; dot plots indicate relative survival/death of ß cells, and histograms compare GCG and INS levels in the respective uninfected, NP-, and NP+ fractions of a or ß cells.All summary diagrams represent mean ± SD and/or scatter for the indicated numbers of donors; statistics were calculated by paired t test or repeated-measures ANOVA with Tukey’s multiple comparisons where applicable (*, p < 0.05; **, p < 0.01).

Fig 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.

Fig 4: LINC00261 deletion impedes pancreatic endocrine cell differentiation.(A) Flow cytometry analysis at endocrine cell stage (EC) for insulin (INS) in control (ctrl) and LINC000261-/- H1 hESCs. Top panel: Schematic of the LINC00261 locus. The dashed box represents the genomic deletion. Middle panel: The line demarks isotype control. Percentage of cells expressing INS is indicated (representative experiment, n = 4 deletion clones generated with independent sgRNAs). Bottom panel: Bar graph showing percentages of INS-positive cells. Data are shown as mean ± S.D. (n = 5 (clone 1), n = 6 (clone 2), n = 8 (clone 3), n = 5 (clone 4) independent differentiations). Individual data points are represented by dots. (B) Immunofluorescence staining for INS in EC stage cultures from control and LINC000261-/- hESCs (representative images, number of differentiations see A). Boxed areas (dashed boxes) are shown in higher magnification. (C) ELISA for INS in EC stage cultures from control and LINC00261-/- hESCs. Data are shown as mean ± S.D. (n = 3 (clone 1), n = 2 (clone 2), n = 14 (clone 3), n = 13 (clone 4) independent differentiations). Individual data points are represented by dots. (D) qRT-PCR analysis of INS in EC stage cultures from control and LINC00261-/- hESCs. Data are shown as mean ± S.E.M. (n = 8 (clone 1), n = 4 (clone 2), n = 10 (clone 3), n = 3 (clone 4) independent differentiations). Individual data points are represented by dots. (E) Quantification of median fluorescence intensity after INS staining of control and LINC00261-/- EC stage cultures. Data are shown as mean ± S.D. (n = 5 (clone 1), n = 5 (clone 2), n = 4 (clone 3), n = 4 (clone 4) independent differentiations). iso, isotype control. Individual data points are represented by dots. (F) Volcano plot displaying gene expression changes in control versus LINC00261-/- PP2 cells (n = 6 independent differentiations from all four deletion clones). Differentially expressed genes are shown in red (DESeq2;>2 fold change (FC), adjusted p-value<0.01) and blue (>2 fold change, adjusted p-value≥0.01 and≤0.05). Thresholds are represented by vertical and horizontal dashed lines. FOXA2 in cis to LINC00261 is shown in gray (gray dots represent genes with ≤ 2 fold change and/or adjusted p-value>0.05). (G) Circos plot visualizing the chromosomal locations of the 108 genes differentially expressed (DESeq2;>2 fold change (FC), adjusted p-value<0.01) in LINC00261-/- compared to control PP2 cells, relative to LINC00261 on chromosome 20. No chromosome was over- or underrepresented (Fisher test, p-value>0.05 for all chromosomes). (H) Top panel: Schematic of the LINC00261 locus, with the location of its sORFs (1 to 7) marked by vertical red bars. Bottom panel: Flow cytometric quantification of INS-positive cells in control and LINC00261-sORF-frameshift (FS) at the EC stage. Data are shown as mean ± S.D. (n = 4–7 independent differentiations per clone). (I) ELISA for INS in EC stage cultures from control and LINC00261-sORF-FS hESCs. Data are shown as mean ± S.D. (n = 3–7 independent differentiations per clone). (J) Volcano plot displaying gene expression changes in control versus LINC00261-sORF3-FS PP2 cells. No gene was differentially expressed (DESeq2;>2 fold change, adjusted p-value<0.01; indicated by dashed horizontal and vertical lines; n = 2 independent differentiations). LINC00261 is shown in gray, the bar graph insert displays LINC00261 RPKM values in control and LINC00261-sORF3-FS PP2 cells. (K) LINC00261 half-life measurements in HEK293T cells transduced with lentivirus expressing either wild type (WT) LINC00261 or ΔATGsORF1-7 LINC00261 (mutant in which the ATG start codons of sORFs 1–7 were changed to non-start codons). HEK293T were treated with the transcription inhibitor actinomycin D and RNA isolated at 0, 2, 4, 6, 8, and 9 hr post actinomycin D addition. LINC00261 expression was analyzed by qRT-PCR relative to the TBP gene. Data are shown as mean ± S.E.M. (n = 3 biological replicates for each assay time point). *, p-value<0.05; **, p-value<0.01; ***, p-value<0.001; ****, p-value<0.0001; NS, p-value>0.05; t-test. Scale bars = 100 µm. See also Figure 4—figure supplement 1 and Figure 4—source data 1–3.Figure 4—source data 1.Characterization of LINC00261 knockout and LINC00261-sORF3-frameshift PP2 cells.(A) Differentially expressed genes in LINC00261 knockout PP2 stage cells. (B) Sequences of LINC00261 wild type and frameshift mutants. (C) Differentially expressed genes in LINC00261-sORF3-frameshift PP2 stage cells.Figure 4—source data 2.List of oligonucleotides and synthetic gene fragments used in this study.(A) sgRNA oligonucleotides used for cloning into PX458/Px459. (B) Genotyping and sequencing primers for KO validation. (C) qRT-PCR primers. (D) Cloning primers (translation reporter constructs and lentiviral LINC00261 overexpression plasmids). (E) Synthetic gene fragments. (F) Custom LINC00261 Stellaris RNA FISH probe set.Figure 4—source data 3.Source data used for the insulin measurements presented in Figure 4.