Mutagenesis:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
Construct:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
Clone Assay:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
Plasmid Preparation:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
Sequencing:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
RNA Sequencing:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
Expressing:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
Migration:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
Activity Assay:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
Virus:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
Methylation:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
shRNA:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
Cloning:Article Title: DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells.
Article Snippet: .. Sox2 silencing in Dnmt3b mutant cells Custom shRNAs against Sox2 were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the following sequences: 5′-ACCAATCCCATCCAAATTAAC-3′ (shRNA1) 5′-GCACAGTTTGAGATAAATAAA-3′ (shRNA2) Hairpins were cloned into pLKO.1 vector (Addgene plasmid_#10878) and each construct was verified by sequencing. a 0 1 D im .2 Clone B126 B77 WT MFA [ RNA-seq + WGBS ] b 3BKO upregulated genes [expression] d GO: Biological Process [top 10] ea rly m id la te microtubule bundle formation axoneme assembly male meiotic nuclear division negative reg. of cell morphogenesis involved in differentiation cilium organization intrinsic apoptotic signaling pathway in response to DNA damage pattern specification process telencephalon cell migration negative reg. of neuron projection development cilium movement import into cell reg. of cation channel activity calcium ion transmembrane transport positive reg.of transporter activity positive reg.of ion transport reg. of transporter activity axonogenesis hindbrain morphogenesis central nervous system neuron differentiation cellular response to virus dendrite development negative reg.of nervous system development reg.of cell morphogenesis involved in differentiation regionalization urogenital system development 0 2 4 6 c m id la te Gli2 Sox2 Nnat Sox1 Id2 Olig3 WGBS Z score (logRPKM) Regulatory Region D iff er en tia lly in du ce d ge ne s Methylation [%] 0 50 100 Promoters SuperEnhancers TypicalEnhancers WT 3BKOWT 3BKO Differentiation [direct targets] Stage-Genotype 1# 2# 1# 2#1# 2#1# 2# B7 7 B1 26 B1 26 B7 7 B1 26 B7 7 B1 26 B7 71# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# 1# 2# 1# 2#1# 2#1# 2# B126 B77 B126 B77 B126 B126B77 B77 ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME 24h ME 48h ESC EpiLC ME24h ME48h ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO Stage-Genotype ESC-WT EpiLC-WT ME24h-WT ME48h-WT ESC-3BKO EpiLC-3BKO ME24h-3BKO ME48h-3BKO 3BKO-B126 3BKO-B77 WT#1 WT#2 A ve ra ge e xp re ss io n [s ca le d R P K M ] A ve ra ge m et hy la tio n [% ] W G B S early mid late ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h ES C Ep iLC M E2 4h M E4 8h 25 50 75 Nature Communications | (2023) 14:367 10 Oligonucleotide sequences for shRNA cloning are reported in Supplementary Data 8. .. For Sox2 silencing, EpiLCs were plated in 6-well plates and the day after they were transfected with 5μg of the specific shRNA construct using Lipofectamine 2000 Transfection Reagent (Invitrogen_#11668500) in accordance with the manufacturer’s protocol, and maintained in meso-endoderm differentiation medium for 48 h. Cells were collected for RNA and protein analyses at 48 h of the meso-endoderm differentiation.
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