Figures and data in A human ESC-based screen identifies a role for the translated lncRNA LINC00261 in pancreatic endocrine differentiation

Figures
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4 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement

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LncRNA expression and regulation during pancreatic differentiation.

(A) Stages of directed differentiation from human embryonic stem cell (hESCs) to hormone-producing endocrine cells. The color scheme for each stage is used across all figures. (B) K-means clustering of all lncRNAs expressed (RPKM ≥ 1) during pancreatic differentiation based on their expression z-score (mean of n = 2 independent differentiations per stage; from CyT49 hESCs). (**C,D**) Left: Scatterplots comparing the expression of early (C) and late (D) expressed endodermal transcription factors (TFs) with the expression of their neighboring lncRNAs across 38 tissues. The dot color indicates the germ layer of origin of these tissues. Pearson correlation coefficients and p-values (t-test) are displayed. Right: Distribution of the Pearson correlation coefficients for each TF with all Ensembl 87 genes across the same 38 tissues. Dashed lines denote the correlation for the neighboring lncRNA, which for all lncRNAs shown is higher than expected by chance. See also Figure 1—figure supplement 1 and Figure 1—source data 1.

Figure 1—source data 1 Identification, regulation, and characterization of lncRNAs during pancreatic differentiation. (A) Gene expression during pancreatic differentiation (RPKM). (B) lncRNA-proximal TFs, by cluster in correlation heatmap (Figure 1—figure supplement 1C). (C) GO enrichment and KEGG pathway analysis for each cluster in the correlation heatmap (Figure 1—figure supplement 1D).: https://cdn.elifesciences.org/articles/58659/elife-58659-fig1-data1-v2.xlsx
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Figure 1—figure supplement 1

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Characterization of lncRNAs expressed during pancreatic differentiation.

(**A,B**) Left: Expression of the single nearest coding genes (±1000 kb) in cis to transcribed and non-transcribed lncRNAs at the DE stage (A) or PP2 stage (B). Log₂ transformed mean expression values (RPKM + pseudocount) from two biological replicates were used to generate the box plots (****, p-value<0.0001, Wilcoxon rank sum test). Right: Corresponding cumulative distance distribution functions. (C) Heatmap of the hierarchically clustered expression correlations (Spearman’s rho) of all RNAs transcribed during pancreatic differentiation (with RPKM ≥ 1 in at least ten out of 38 tissues). Transcription factor (TF)-encoding mRNAs, lncRNAs (all), dynamically expressed lncRNAs (RPKM ≤ 1 in at least one stage (ESC to PP2)), and TF-proximal lncRNAs are highlighted above the heatmap. Clusters 8 and 10 are significantly enriched for all of these RNAs (*, p-value<0.03, Fisher test). (D) Gene ontology and KEGG pathway analysis for all coding genes in cluster 8 (p-value<0.05, Fisher test). The full list of significantly enriched terms is shown in Figure 1—source data 1C. (**E–H**) H3K4me3 and H3K27me3 ChIP-seq tracks of loci containing lncRNAs *GATA6-AS1* (A), *LINC00261* (B), *PDX1-*AS1/*PLUTO* (C), or *SOX9-AS1* (D) during pancreatic differentiation of CyT49 hESCs.

Figure 2 with 1 supplement

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Cytosolic lncRNAs contain translated small open reading frames.

(A) Overview of experimental strategy for subcellular fractionation and Ribo-seq-based identification of translated small open reading frames (sORFs) from lncRNAs expressed in PP2 cells. Replicates from six independent differentiations to PP2 stage each for total (polyA) RNA-seq and Ribo-seq experiments, and two biological replicates for the subcellular fractionation were analyzed. The histogram on the far right depicts the size distribution of the sORF-encoded small peptides as number of amino acids (aa). The pie chart summarizes the percentages of constitutively and dynamically expressed sORF-encoding lncRNAs during pancreatic differentiation of CyT49 hESCs. (**B–E**) Left: Bar graphs showing nuclear and cytosolic expression (in RPKM) of lncRNAs *RP11-834C11.4* (B), *LINC00261* (C), *MIR7-3HG* (D), and *LHFPL3-AS2* (E). Data are shown as mean, with individual data points represented by dots (n = 2 biological replicates). Right: Subcellular fractionation RNA-seq, Ribo-seq, and P-site tracks (ribosomal P-sites inferred from ribosome footprints on ribosome-protected RNA) for loci of the depicted lncRNAs. Identified highest stringency sORFs (ORF in 6/6 replicates) are shown in red. For *LINC00261*, visually identified sORFs 1 and 2 are also shown. Heatmaps in the top right visualize the relative expression of the shown lncRNAs during pancreatic differentiation (means of two biological replicates per stage), on a minimum (white)/maximum (dark blue) scale. (F) In vivo translation reporter assays testing whether sORFs computationally defined in (A) give rise to translation products in HEK293T cells when fused in-frame to a GFP reporter. Left: Schematic of the constructs (gray: *PGK* promoter, black: lncRNA sequence 5’ to sORF to be tested, red: sORF, green: GFP ORF). Right: Representative DIC and GFP images of HEK293T cells transiently transfected with the indicated reporter constructs. Scale bars = 50 µm. See also Figure 2—figure supplement 1 and Figure 2—source data 1.

Figure 2—source data 1 RNA-seq after subcellular fractionation and Ribo-seq in PP2 cells. (A) Subcellular fractionation of PP2 stage cells (RPKM). (B) Ribo-seq/mRNA-seq contaminant filtering statistics, read size distribution, and Pearson correlation coefficients of all sequenced Ribo-seq and polyA RNA-seq libraries. (C) All ORFs detected by RiboTaper, including lncRNA sORFs. (D) lncRNA sORFs detected by RiboTaper and conservation statistics (PhyloCSF scores). (E) Translational efficiency calculations.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig2-data1-v2.xlsx
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Figure 2—figure supplement 1

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Cytosolic lncRNAs engage with ribosomes.

(A) Venn diagrams showing the number of coding RNAs (left) and lncRNAs (right) with RPKM ≥ 1 across two biological replicates in cytosolic and nuclear fractions. (B) Box plots of maximum lncRNA expression (RPKM + pseudocount) across 38 tissues binned by their degree of cytosolic localization (measured as nuclear/cytosolic lncRNA expression ratio deciles in PP2); the expression of all PP2-transcribed coding RNAs with dynamic expression during differentiation is included for reference. The pie chart summarizes the proportions of translated lncRNAs within each cytoplasmic localization decile. (C) Read length distribution (nt) of Ribo-seq fragments across replicate Ribo-seq experiments (n = 6 biological replicates). (D) Position of the inferred P-sites of the ribosome footprints relative to the reading frame of PP2-transcribed coding genes. (**E–F**) Coverage of 29 nt footprint P-sites around the start codons (E) or stop codons (F) of PP2-transcribed coding genes. (G) Box plots comparing maximum expression of translated and untranslated lncRNAs (RPKM + pseudocount) across 38 tissues (****, p-value=2.122×10⁻⁸, Wilcoxon rank sum test). For the untranslated set, 285 untranslated PP2-expressed lncRNAs were selected randomly. (H) Density plots comparing the translation efficiencies of PP2-expressed mRNAs and lncRNAs. (I) Autoradiograph of radiolabeled in vitro translation products derived from full-length *LHFPL3-AS2*, *MIR7-3HG*, *LINC00261*, and *RP11-834C11.4*. EV, empty vector. (J) Anti-FLAG immunofluorescence staining of HEK293T cells transiently transfected with a *PGK-RP11-834C11.4*-sORF-1xFLAG construct. (K) Microphotograph of HEK293T cells transiently transfected with a *PGK-LINC00261*-sORF4-*GFP* construct with mitochondria labeled by MitoSOX Red. (L) Golgi immunofluorescence staining (anti-GM130) of HEK293T cells transiently transfected with a *PGK-LINC00261*-sORF7-*GFP* construct. Scale bars = 10 µm.

Figure 3 with 1 supplement

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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.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig3-data1-v2.xlsx
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Figure 3—source data 2 Source data used for the qRT-PCR quantification of gene expression presented in Figure 3A.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig3-data2-v2.xlsx
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Figure 3—source data 3 Source data used for the qRT-PCR quantification of gene expression presented in Figure 3D.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig3-data3-v2.xlsx
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Figure 3—source data 4 Source data used for the qRT-PCR quantification of INS expression presented in Figure 3H.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig3-data4-v2.xlsx
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Figure 3—figure supplement 1

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Minor gene expression changes in definitive endoderm or pancreatic progenitor cells after lncRNA deletion.

(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).

Figure 4 with 1 supplement

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*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 ΔATG^sORF1-7 *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.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig4-data1-v2.xlsx
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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.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig4-data2-v2.xlsx
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Figure 4—source data 3 Source data used for the insulin measurements presented in Figure 4.: https://cdn.elifesciences.org/articles/58659/elife-58659-fig4-data3-v2.xlsx
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Figure 4—figure supplement 1

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Characterization of *LINC00261*-deleted pancreatic progenitor cells.

(A) RNA-seq expression heatmap of *LINC00261* across 35 cell types/tissues originating from all three germ layers (shown as RPKM + pseudocount). (B) Heatmap showing K-means clustering of 108 differentially expressed genes (DESeq2;>2 fold change (FC), adjusted p-value<0.01) between PP2 cells from control and *LINC00261^-/-* H1 hESCs (based on expression z-score; n = 6 independent differentiations). (C) Top: Genome Browser snap shot of RNA-seq signal at the *LINC00261*/*FOXA2* locus in control and *LINC00261^-/-* PP2 stage cells. Genomic deletions are indicated by gray boxes. Bottom: Bar graphs showing *LINC00261* and *FOXA2* expression in control and *LINC00261^-/-* PP2 cells quantified by RNA-seq. Data are shown as mean RPKM ± S.D. (n = 6 independent differentiations of four independent KO clones). ****, p-value<0.0001; NS, p-value>0.05; t-test. (D) *LINC00261* smRNA FISH in control and *LINC00261^-/-* PP2 cells. Scale bars = 8 µm. Boxed areas (dashed boxes) are shown in higher magnification. (E) qRT-PCR analysis of *LINC00261* (top) and *INS* (bottom) expression in control and *LINC00261*-sORF-FS H1 hESC clones at the endocrine cell (EC) stage. Data are shown as mean ± S.E.M. (n ≥ 3 independent differentiations for each clone). Individual data points are represented by dots.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Homo sapiens)	LINC00617; TUNAR	Ensembl 87	ENSG00000250366
Gene (Homo sapiens)	RP11-445F12.1; LHX-DT	Ensembl 87	ENSG00000250366
Gene (Homo sapiens)	DIGIT; GSC-DT	HGNC and NCBI RefSeq	HGNC:53074; NCBI RefSeq 108868751
Gene (Homo sapiens)	GATA6-AS1; GATA6-AS	Ensembl 87	ENSG00000277268
Gene (Homo sapiens)	LINC00479	Ensembl 87	ENSG00000236384
Gene (Homo sapiens)	LINC00261; DEANR1; ALIEN; onco-lncRNA-17; lnc-FOXA2-2	Ensembl 87	ENSG00000236384
Gene (Homo sapiens)	RP11-834C11.4	Ensembl 87	ENSG00000250742
Gene (Homo sapiens)	SOX9-AS1	Ensembl 87	ENSG00000234899
Gene (Homo sapiens)	MIR7-3HG	Ensembl 87	ENSG00000176840
Gene (Homo sapiens)	LHFPL3-AS2	Ensembl 87	ENSG00000225329
Strain, strain background (Escherichia coli)	Stbl3	ThermoFisher Scientific	Cat# C737303	Chemically competent cells
Strain, strain background (Escherichia coli)	DH5α	New England Biolabs	Cat# C2987I	Chemically competent cells
Cell line (Homo sapiens)	H1 (embryonic stem cells)	WiCell Research Institute	NIHhESC-10–0043, RRID:CVCL_9771
Cell line (Homo sapiens)	HEK293T (embryonic kidney)	ATCC	Cat# CRL-3216, RRID:CVCL_0063
Antibody	anti-human OCT-4A (Rabbit monoclonal)	Cell Signaling Technology	Cat# 2890, RRID:AB_2167725	IF (1:1000)
Antibody	anti-human SOX17 (Goat polyclonal)	R and D Systems	Cat# AF1924, RRID:AB_355060	IF (1:250)
Antibody	anti-human FOXA2 (Goat polyclonal)	Santa Cruz Biotechnology	Cat# sc-6554, RRID:AB_2262810	IF (1:250)
Antibody	anti-human GATA6 (Goat polyclonal)	Santa Cruz Biotechnology	Cat# sc-9055, RRID:AB_2108768	IF (1:50)
Antibody	anti-human Insulin (Guinea pig polyclonal)	Dako	Cat# A0564, RRID:AB_10013624	IF (1:1000)
Antibody	Alexa Fluor 488 AffiniPure anti-Goat IgG (Donkey polyclonal)	Jackson ImmunoResearch Labs	Cat# 706-545-148, RRID:AB_2340472	IF (1:1000)
Antibody	Alexa Fluor 488 AffiniPure anti-Rabbit IgG (Donkey polyclonal)	Jackson ImmunoResearch Labs	Cat# 711-545-152, RRID:AB_2313584	IF (1:1000)
Antibody	Cy3 AffiniPure anti-Goat IgG (Donkey polyclonal)	Jackson ImmunoResearch Labs	Cat# 705-165-147, RRID:AB_2307351	IF (1:1000)
Antibody	anti-human Insulin-PE (Rabbit monoclonal)	Cell Signaling Technology	Cat# 8508S, RRID:AB_11179076	Flow cytometry (1:50)
Antibody	anti-human SOX17-PE (Mouse monoclonal)	BD Biosciences	Cat# 561591, RRID:AB_10717121	Flow cytometry (5 ul per test)
Antibody	IgG-PE (Rabbit monoclonal)	Cell Signaling Technology	Cat# 5742S, RRID:AB_10694219	Flow cytometry isotype control antibody (1:50)
Antibody	IgG1, κ antibody (Mouse monoclonal)	BD Biosciences	Cat# 556650, RRID:AB_396514	Flow cytometry isotype control antibody (1:50)
Recombinant DNA reagent	pSpCas9(BB)−2A-Puro (Px459; V2.0)	Feng Zhang	RRID:Addgene_62988	Cas9 from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA
Recombinant DNA reagent	pSpCas9(BB)−2 A-GFP (PX458)	Feng Zhang	RRID:Addgene_48138	Cas9 from S. pyogenes with 2A-EGFP, and cloning backbone for sgRNA
Recombinant DNA reagent	pCMVR8.74	Didier Trono	RRID:Addgene_22036	2nd generation lentiviral packaging plasmid
Recombinant DNA reagent	pMD2.G	Didier Trono	RRID:Addgene_12259	VSV-G envelope expressing plasmid
Recombinant DNA reagent	pENTR/D-TOPO-LINC00261	Kurian et al., 2015 (PMID:25739401)		Leo Kurian (University of Cologne)
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-GFP.WPRE	Didier Trono	RRID:Addgene_12252	3rd generation lentiviral backbone
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of wild type LINC00261
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-ΔATG^sORF1-7 LINC00261.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of ΔATG^sORF1-7 LINC00261
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF1-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF1-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF2-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF2-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF3-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF3-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF4-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF4-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF5-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF5-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF6-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF6-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF7-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LINC00261-sORF7-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF3-FS-GFP.WPRE	This paper		LINC00261-sORF3-frameshift-GFP control plasmid
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-LINC00261-sORF2-STOP-GFP.WPRE	This paper		LINC00261-sORF2-STOP-GFP control plasmid
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-RP11-834C11.4-sORF-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of RP11-834C11.4-sORF-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-RP11-834C11.4-sORF-FLAG.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of RP11-834C11.4-sORF-FLAG fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-MIR7-3HG-sORF-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of MIR7-3HG-sORF-GFP fusion protein
Recombinant DNA reagent	pRRLSIN.cPPT.PGK-RP11-LHFPL3-AS2-sORF-GFP.WPRE	This paper		Transient (transfection) or stable (lentiviral integration) expression of LHFPL3-AS2-sORF-GFP fusion protein
Peptide, recombinant protein	Activin A	R and D Systems	Cat# 338-AC/CF
Peptide, recombinant protein	Wnt3a	R and D Systems	Cat# 1324-WN-010
Peptide, recombinant protein	KGF/FGF7	R and D Systems	Cat# 251 KG
Peptide, recombinant protein	Noggin	R and D Systems	Cat# 3344 NG-050
Commercial assay or kit	RNeasy Mini Kit	QIAGEN	Cat# 15596018
Commercial assay or kit	RNA Clean and Concentrator−25	Zymo Research	Cat# R1018
Commercial assay or kit	Paris Kit	Thermo Fisher Scientific	Cat# AM1921
Commercial assay or kit	RNase-Free DNase Set (50)	QIAGEN	Cat# 79254
Commercial assay or kit	TURBO DNA-free Kit	Thermo Fisher Scientific	Cat# AM1907
Commercial assay or kit	TruSeq Ribo Profile (Mammalian) Library Prep Kit	Illumina	Cat# RPYSC12116
Commercial assay or kit	TruSeq Stranded mRNA Library Prep	Illumina	Cat# 20020594
Commercial assay or kit	TruSeq Stranded Total RNA Library Prep Gold	Illumina	Cat# 20020599
Commercial assay or kit	KAPA mRNA HyperPrep Kit	Roche	Cat# KK8581
Commercial assay or kit	High Sensitivity D1000 ScreenTape	Agilent Technologies	Cat# 5067–5584
Commercial assay or kit	RNA ScreenTape	Agilent Technologies	Cat# 5067–5576
Commercial assay or kit	RNA ScreenTape Sample Buffer	Agilent Technologies	Cat# 5067–5577
Commercial assay or kit	RNA ScreenTape Ladder	Agilent Technologies	Cat# 5067–5578
Commercial assay or kit	Qubit ssDNA assay kit	Thermo Fisher Scientific	Cat# Q10212
Commercial assay or kit	KOD Xtreme DNA Hotstart Polymerase	Millipore	Cat# 71975
Commercial assay or kit	GoTaq Green Master Mix	Promega	Cat# M7123
Commercial assay or kit	TOPO TA Cloning Kit	Thermo Fisher Scientific	Cat# K452001
Commercial assay or kit	Monarch Plasmid Miniprep Kit	NEB	Cat# T1010L
Commercial assay or kit	MinElute PCR Purification Kit	QIAGEN	Cat# 28006
Commercial assay or kit	iScript cDNA Synthesis Kit	Bio-Rad	Cat# 1708890
Commercial assay or kit	iQ SYBR Green Supermix	Bio-Rad	Cat# 1708880
Commercial assay or kit	Human Stem Cell Nucleofector Kit 2	Lonza	Cat# VPH-5022
Commercial assay or kit	XtremeGene 9 DNA Transfection Reagent	Sigma-Aldrich	Cat# 06365779001
Commercial assay or kit	Cytofix/Cytoperm W/Golgi Stop Kit	BD Biosciences	Cat# 554715
Commercial assay or kit	Insulin ELISA Jumbo	Alpco	Cat# 80-INSHU-E10.1
Commercial assay or kit	Pierce BCA Protein Assay Kit	Thermo Fisher Scientific	Cat# 23227
Commercial assay or kit	TnT Coupled Wheat Germ Extract System	Promega	Cat# L4130
Chemical compound, drug	Penicillin-Streptomycin	Thermo Fisher Scientific	Cat# 15140122
Chemical compound, drug	Puromycin dihydrochloride	Sigma-Aldrich	Cat# P8833
Chemical compound, drug	ALK5 Inhibitor II	Enzo Life Sciences	Cat# ALX-270–445
Chemical compound, drug	Retinoic Acid	Sigma-Aldrich	Cat# R2625
Chemical compound, drug	Ascorbic Acid	Sigma-Aldrich	Cat# A4403-100MG
Chemical compound, drug	LDN-193189	Stemgent	Cat# 04–0074
Chemical compound, drug	SANT-1	Sigma-Aldrich	Cat# S4572
Chemical compound, drug	TPB	Calbiochem	Cat# 565740
Chemical compound, drug	TGFβ R1 kinase inhibitor IV	EMD Biosciences	Cat# 616454
Chemical compound, drug	KAAD-Cyclopamine	Toronto Research Chemicals	Cat# K171000
Chemical compound, drug	TTNPB	Sigma-Aldrich	Cat# T3757
Chemical compound, drug	Cycloheximide	Sigma-Aldrich	Cat# C7698
Chemical compound, drug	Actinomycin D	Sigma-Aldrich	Cat# A9415
Chemical compound, drug	Polyethylenimine (PEI)	Polysciences	Cat# 23966–1
Chemical compound, drug	Hoechst 33342, Trihydrochloride, Trihydrate	Thermo Fisher Scientific	Cat# H3570
Chemical compound, drug	MitoSOX Red	Thermo Fisher Scientific	Cat# M36008
Chemical compound, drug	D-(+)-Glucose Solution, 45%	Sigma-Aldrich	Cat# G8769
Chemical compound, drug	Sodium Bicarbonate	Sigma-Aldrich	Cat# NC0564699
Chemical compound, drug	ROCK Inhibitor Y-27632	STEMCELL Technologies	Cat# 72305
Software, algorithm	Flowjo-v10	FlowJo, LLC	RRID:SCR_008520	http://www.flowjo.com/download-newest-version/
Software, algorithm	STAR 2.5.3a	Dobin et al., 2013	RRID:SCR_015899	https://github.com/alexdobin/STAR
Software, algorithm	Bowtie 1.1.1	Langmead et al., 2009	RRID:SCR_005476	http://bowtie-bio.sourceforge.net/index.shtml
Software, algorithm	Cufflinks 2.2.1	Trapnell et al., 2010	RRID:SCR_014597	https://github.com/cole-trapnell-lab/cufflinks
Software, algorithm	HTSeq 0.6.1	Anders et al., 2015	RRID:SCR_005514	https://htseq.readthedocs.io/en/master/install.html
Software, algorithm	DEseq2 1.10.1	Love et al., 2014	RRID:SCR_015687	https://www.bioconductor.org/packages/devel/bioc/html/DESeq2.html
Software, algorithm	RiboTaper	Calviello et al., 2016	RRID:SCR_018880	https://ohlerlab.mdc-berlin.de/software/RiboTaper_126/
Software, algorithm	R 3.5.0		RRID:SCR_001905	https://cran.r-project.org/
Software, algorithm	SAMtools 1.3	Li et al., 2009	RRID:SCR_002105	https://github.com/samtools/samtools
Software, algorithm	BEDTools 2.17.0	Quinlan and Hall, 2010	RRID:SCR_006646	https://bedtools.readthedocs.io/en/latest/content/installation.html
Software, algorithm	HOMER 4.10	Heinz et al., 2010	RRID:SCR_010881	http://homer.ucsd.edu/homer/download.html
Software, algorithm	GREAT 3.0.0	McLean et al., 2010	RRID:SCR_005807	http://great.stanford.edu/public/html/
Software, algorithm	Adobe Illustrator CS5	Adobe	RRID:SCR_010279
Software, algorithm	Adobe Photoshop CS5	Adobe	RRID:SCR_014199
Software, algorithm	GraphPad Prism v7.05	GraphPad Software, LLC	RRID:SCR_002798
Other	Novex 16% Tricine Protein Gel	Thermo Fisher Scientific	Cat# EC66955BOX
Other	Novex Tricine SDS Sample Buffer (2X)	Thermo Fisher Scientific	Cat# LC1676
Other	Immobilon-PSQ PVDF Membrane	Merck Millipore	Cat# ISEQ00010
Other	Stellaris RNA FISH Hybridisation Buffer	LGC Biosearch Technologies	Cat# SMF-HB1-10
Other	Stellaris RNA FISH Wash Buffer A	LGC Biosearch Technologies	Cat# SMF-WA1-60
Other	Stellaris RNA FISH Wash Buffer B	LGC Biosearch Technologies	Cat# SMF-WB1-20
Other	QuickExtract DNA Extraction Solution	Lucigen	Cat# QE09050
Other	Vectashield Antifade Mounting Medium	Vector Laboratories	Cat# H-1000
Other	FastDigest BpiI	Thermo Fisher Scientific	Cat# FD1014
Other	FastDigest BshTI	Thermo Fisher Scientific	Cat# FERFD1464
Other	FastDigest SalI	Thermo Fisher Scientific	Cat# FD0644
Other	TRIzol	Thermo Fisher Scientific	Cat# 15596018
Other	Matrigel	Corning	Cat# 356231
Other	GlutaMAX	Thermo Fisher Scientific	Cat# 35050061
Other	DPBS (without calcium and magnesium)	Corning	Cat# 45000–434
Other	mTeSR1 Complete Kit - GMP	STEMCELL Technologies	Cat# 85850
Other	RPMI 1640 Medium, HEPES	Thermo Fisher Scientific	Cat# 22400–089
Other	DMEM/F12 with L-Glutamine, HEPES	Corning	Cat# 45000–350
Other	Dulbecco’s Modified Eagle’s Medium	Corning	Cat# 45000–312
Other	HyClone Dulbecco’s Modified Eagles Medium	Thermo Fisher Scientific	Cat# SH30081.FS
Other	MCDB 131	Thermo Fisher Scientific	Cat# 10372–019
Other	Opti-MEM Reduced Serum Medium	Thermo Fisher Scientific	Cat# 31985062
Other	Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) (100X)	Thermo Fisher Scientific	Cat# 51500–056
Other	B-27 Supplement (50X)	Thermo Fisher Scientific	Cat# 17504044
Other	Bovine Albumin Fraction V (7.5%)	Thermo Fisher Scientific	Cat# 15260037
Other	Fetal Bovine Serum	Corning	Cat# 35011CV
Other	Donkey Serum	Gemini Bio-Products	Cat# 100-151/500
Other	Fatty Acid-Free BSA	Proliant Biologicals	Cat# 68700
Other	Accutase	eBioscience	Cat# 00-4555-56