GSM8358744: WT_E15.5_728; Mus musculus; RNA-Seq
For Smart-seq2 scRNA-seq, single cells were manually picked from FACS-purified bulk cells using a mouth pipette. cDNA synthesis and amplification were performed following the protocol by Picelli et al. For Smart-seq3 scRNA-seq, single cells were directly sorted into 384-well plates containing lysis buffer. cDNA synthesis and amplification were carried out according to the Smart-seq3 protocol .For 10x Genomics scRNA-seq, the procedure was performed according to the manufacturer's instructions using the Single Cell 3′ Reagent Kit v3.1. Sorted EpCAM+ pancreatic cells from wild-type (WT), conditional knockout (CKO), or overexpression (OE) embryos of different genders were pooled and processed as one sample. For NGN3-CKO samples, E14.5 EpCAM+ pancreatic cells were pooled per replicate: replicate 1 consisted of two WT male litters and one CKO female litter, and replicate 2 consisted of three WT male litters and three CKO female litters. For NGN3-OE samples, E14.5 EpCAM+ pancreatic cells were pooled from four WT female litters and four OE male litters, and E17.5 EpCAM+ pancreatic cells were pooled from two WT female litters and three OE male litters. For NeuroD1-CKO samples, E15.5 EpCAM+ pancreatic cells were pooled per replicate: replicate 1 consisted of one WT male litter and one CKO female litter, and replicate 2 consisted of two WT female litters and one CKO male litter. The gender of the embryos was determined by genotyping for Y-chromosome-associated gene Sry. For Smart-seq2 scRNA-seq, libraries were prepared with 2 ng of cDNA using the TruePrep DNA Library Prep Kit (Vazyme, TD502), and sequenced on the Illumina HiSeq 4000 system, generating 150 bp paired-end reads. For Smart-seq3 scRNA-seq, libraries were constructed using the TruePrep DNA Library Prep Kit V2 for lllumina (Vazyme, TD501), and sequencing was conducted on the MGIseq-2000 platform, generating 100 bp paired-end reads. For 10x Genomics scRNA-seq, libraries were sequenced as 150-bp paired-end reads on an Illumina HiSeq 4000 system. CUT&RUN was conducted following the published protocol with modifications in DNA purification and library construction steps. We used 30,000 cells per CUR&RUN assay with primary antibodies against FLAG (1:25, CST, 2368S), p300 (1;100, CST, 54062S), and NeuroD1 (1:100, Millpore, ABE991). DNA was purified using a spin column (VISTECH, DC2005) and processed for library construction with KAPA Hyper Prep Kit (KAPA biosystems, KK8502). Briefly, after end repair and adaptor ligation, the reaction was purified using VAHTS DNA Clean Beads (Vazyme, N411-03-AA) and subjected to PCR amplification with 2× KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KM2602) and Illumina universal primers. The PCR program was: 98°C for 45 seconds, followed by 19 cycles of 98°C for 15 seconds, 60°C for 10 seconds, and 72°C for 1 minute, with a final extension at 72°C for 5 minutes. The final DNA was purified using 1× VAHTS DNA Clean Beads. CUT&RUN libraries were subjected to 150 bp paired-end sequencing or 51 bp single-end sequencing on the Illumina NovaSeq 6000 or Illumina Hiseq 2500 platform. Bulk-cell ATAC-seq was conducted following the published protocol  with modifications in cell sorting and nuclei preparation. Specifically, 2,000 cells per ATAC-seq were directly sorted into 50 ul lysis buffer in a 200 ul tube for nuclei preparation. Libraries were constructed using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD502). PCR amplification was performed for 12 cycles under the following conditions: 72°C for 3 minutes, 98°C for 30 seconds, then 12 cycles of 98°C for 15 seconds, 60°C for 30 seconds, and 72°C for 3 minutes, followed by a final extension at 72°C for 5 minutes. The final DNA was purified using 1× VAHTS DNA Clean Beads and sequenced as 150 bp paired-end reads on the Illumina NovaSeq 6000 platform. For snATAC-seq, the procedure was performed according to the manufacturer's instructions using the Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit v1.1 (10x Genomics). For the Ngn3-CKO samples, E14.5 EpCAM+ pancreatic cells of WT and CKO embryos with different genders (two WT male litters and two CKO female litters) were pooled and loaded as one sample. To ensure experimental success and an adequate number of nuclei, extra pancreatic cells from WT embryos were included, with a ratio of to EpCAM+ cells of 1:4. For the NeuroD1-CKO samples, E15.5 EpCAM+ pancreatic cells of WT and CKO embryos with different genders (two WT female litters and three CKO male litters) were pooled and loaded as one sample. Libraries were sequenced as 50-bp paired-end reads on an NovaSeq 6000 system. snATAC-seq