grna sequences for eomes knockdown  (GenScript corporation)

Journal: Nature Communications

Article Title: Epigenetic and transcriptional regulations prime cell fate before division during human pluripotent stem cell differentiation

doi: 10.1038/s41467-023-36116-9

Figure Lengend Snippet: a Schematic representation of the experimental plan to characterise the functional relevance of MEK1/2, JNK and p38 pathways in endoderm differentiation. hPSCs were grown for 3 days in culture conditions inducing endoderm differentiation in the presence of small-molecule inhibitors. b QPCR, c FACS and d immunostaining were performed after 3 days for pluripotency markers (POU5F1/OCT4, NANOG and SOX2), mesendoderm/mesoderm markers (BRACHYURY/T, NKX2.5, CDX2 and MIXL1) and endoderm markers (SOX17, EOMES, GATA4 and FOXA2). For QPCR, the boxes show the interquartile range, with the median marked as a heavy horizontal band. Whiskers represent the highest (lowest) datapoint within 1.5 times the interquartile range of the 75th (25th) percentile. The diamonds represent each datapoint. For FACS, one-way ANOVA was performed, followed by Dunnett’s multiple comparisons test where each of the three treatment conditions was compared against the control in n = 5 experiments. The statistical significance for FACS marks adjusted P values: ** (0.0096), **** (<0.0001). For immunostaining, the scale bar represents 200 µm. Experiments represent three replicates. Statistical analysis was performed by two-way ANOVA with multiple comparisons and **** marks adjusted P value <0.0001. e Consensus binding motifs for AP-1 transcription factors (JUN and FOSL2), SMAD2/3 and SMAD4 were obtained from ATAC-seq analyses at 36 h time point with corresponding P values. f The switching of transcription factor complexes during hPSC differentiation to definitive endoderm. SMAD2/3 was immunoprecipitated from nuclear extracts of undifferentiated hPSCs, at 36 and at 72 h after initiating endoderm differentiation and analysed for the co-immunoprecipitation of NANOG, EOMES, GATA4, FOSL2 and JUN. g Schematic representation of the experimental outline for analysing the impact of p38-MAPK and TGFβ/Activin A signalling on SMAD2/3 and FOSL2/JUN binding and H3K27ac enrichment on mesendoderm at 24 h, and endoderm or mesoderm loci at 36 h by ChIP-qPCR. Cells were treated with p38-MAPK and TGFβ/Activin A inhibitors for 12 h in the presence of differentiation signals before sample collection. h FOSL2/JUN and SMAD2/3 cooperative binding to MIXL1 and EOMES regulatory regions at mesendoderm differentiation stage during hPSC differentiation. Early G1 phase synchronised cells were differentiated to endoderm for 12 h to receive the pluripotency exit signalling, and then treated with p38-MAPK inhibitor SB203580 or TGFβ/Activin signalling inhibitor SB431542 for 12 h, followed by cell fixation for ChIP-qPCR (before first cell division). Analyses reveal that p38-MAPK and TGFβ/Activin signalling regulate the cooperative binding of AP-1 TFs FOSL2/JUN and SMAD2/3 to mesendoderm genes at 24 h time point; n = 3 biologically independent experiments. i FOSL2/JUN and SMAD2/3 cooperative binding to CER1 , FOXA2 , LZTS1 , GATA4 and GSC regulatory regions at endoderm differentiation stage during hPSC differentiation. Early G1 phase synchronised cells were differentiated to endoderm for 24 h, and then treated with p38-MAPK inhibitor SB203580 or TGFβ/Activin signalling inhibitor for 12 h, followed by cell fixation for ChIP-qPCR (before second cell division). p38-MAPK and TGFβ/Activin signalling regulate the cooperative binding of AP-1 TFs FOSL2/JUN and SMAD2/3 to definitive endoderm genes at 36 h time point. n = 3 biologically independent experiments. j – m Activation of p38-MAPK signalling improves definitive endoderm and pancreatic differentiation. Cells were treated with 200 mM Sorbitol during 24 to 72 h of endoderm differentiation and analysed by j qPCR of FOXA2 and GSC expression at day 3, k , l FACS of PDX1, SOX9 and CD142 co-expression at day 12, m qPCR of NGN3 , SST , GSG and Insulin expression at day 18 of pancreatic differentiation. Experiments represent three replicates. n Activation of p38-MAPK signalling by Sorbitol improves definitive endoderm differentiation and the formation of endoderm-derived pancreatic cell types. Schematic representation of hESC and hiPSC differentiation to definitive endoderm, pancreatic progenitors and pancreatic islets of the Langerhans that contain ɑ, β, δ and F cells expressing their corresponding secreted factors such as insulin by β cells. Activation of p38-MAPK by Sorbitol improved definitive endoderm differentiation and the formation of subsequent pancreatic cells. Adapted from 'Pancreatic Islet of Langerhans', by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates . Statistical analyses in ( h , i , j , m ) were performed by two-way ANOVA with Tukey’s multiple comparisons tests and * marks adjusted P value <0.05 and ** marks adjusted P value <0.01, *** marks adjusted P value <0.001, **** marks adjusted P value <0.0001. Data were presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Inducible knockdown of EOMES was performed by using a dox-inducible CRISPR interference (CRISPRi) knock-in construct that was first targeted to the AAVS1 locus to create a stable hESC line as described in ref. . gRNA sequences for EOMES knockdown were obtained from the GenScript gRNA database and cloned into the gRNA construct according to the protocol from ref. . 2 mM Doxycyclin (Dox) was used for dCas9-KRAB mediated inducible knockdown of EOMES during endoderm differentiation.

Techniques: Functional Assay, Immunostaining, Control, Binding Assay, Immunoprecipitation, ChIP-qPCR, Activation Assay, Expressing, Derivative Assay