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Status |
Public on Jul 31, 2022 |
Title |
Instructed ESC- H3K27Ac_rep1 |
Sample type |
SRA |
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Source name |
Instructed ESC
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Organism |
Mus musculus |
Characteristics |
sample type: Differentiated ESC
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Treatment protocol |
ESCs differentiation was induced as described inChalet al., 2015. ESC were plated on gelatin-coated plates in serum-free N2B27 medium and BMP4 10ng/ml for 2 days. cells were shifted to MEM, 1%FBS, 14% KSR,10ng/ml Rspo3, 0.5% DMSO and 0.1 μM LDN19318 for 4 days. Subsequently, medium was changed to DMEM, 1%FBS, 14% KSR, 10 ng/ml Rspo3, 10 ng/ml HGF,2 ng/ml IGF-1,20 ng/ml FGF-2 and 0.1 μM LDN193189 for additional 2 days..
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Growth protocol |
ESCs were plated on 0.1% gelatin and cultured in DMEM supplemented with 15% FBS, 1% penicillin-streptomycin,2 mM L-glutamine, 0.1 mM non-essential amino acids, 0.1% β-mercaptoethanol, 1,500 U/ml LIF and 2i inhibitors. Somites were dissected from embryoes, sorted and processed for RNAseq and ATACseq assays.
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Extracted molecule |
genomic DNA |
Extraction protocol |
RNA was extracted using Trizol. For ChIP cells were lysed in RIPA buffer as described in Mousavi et al., 2012. ATAC-seq was performed according to a published protocol (Buenrostro et al., 2013) For RNA-seq, poly (A)+ mRNA libraries were generated using NEBNext Ultra II RNA library preparation kit for Illumina (NEB #E7490). Single-cell RNA-seq libraries were prepared using a Chromium Single Cell 3’ Library & Gel Bead Kit v3.1 (P/N 1000121, 10x Genomics) and scATAC seq performed using Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit v1.1 (10x genomics, 1000175). Single Cell Multiome ATAC+ Gene Expression (10x Genomics kit, CG000338). Hi-C experiments were performed using the Arima-HiC kit (A510008GFP) Bulk RNAseq, Bulk ATAC seq, ChIPseq, scRNA, scATAC, snRNA and scATAC from the same cell (multiomeic) and HiC
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
ChIP-seq
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Data processing |
ChIP-seq reads were mapped to the mm10 genome assembly using Bowtie version 1.1.1,allowing two mismatches, after adapter sequences were removed using trimgalore version0.6.6. RNA-seq reads were mapped to the mm10 genome assembly using TopHat version 2.1.1 with the default configurations after adapter sequences were removed using trimgalore version 0.6.6. peaks were called using MACS 1.4.2 (Zhang et al., 2008) with the following setting: a p value (1.0E-05). Gene expression values (RPKM: Reads Per Kilobase exon per Million mapped reads) were calculated using Partek Genomics Suite 7.18. scRNA-seq reads were processed with CellRanger ver. 3.1.0 and bcl2fastq/2.20.0 to generate fastq files with default parameters. scATAC-seq reads were processed with Cellranger-atac ver. 1.1.0 and bcl2fastq/2.20.0 to generate fastq files with default parameters. omics reads were processed with cellranger-arc/2.0.0 and bcl2fastq/2.20.0 to generate fastq files with default parameters. For clustering, filtering, variable gene selection, dimensionality reduction of scRNA(single cell RNA) are performed using the Seurat ver.4.1.0 (Butler et al., 2018). For clustering, filtering, variable gene selection, dimensionality reduction of scATAC(single cell ATAC) are performed using the Signac ver.1.5.0 (Stuart et al., 2019). The pseudotemporal Analyses of scRNA(single cell RNA) was performed using Monocle v.3.0.0, Slingshot(ver 2.2.0) (Street et al., 2018) and tradeSeq(ver 1.8.0) (Van den Berge et al., 2020). Hi-C reads were mapped to the mm10 genome assembly using BWA (Li and Durbin, 2010),filtering reads that aligned to more than two places in the genome and low mapping quality score (MAPQ <30). Hi-C downstream analysis was performed with HOMER (Heinz et al., 2010) Assembly: mm10 Supplementary files format and content: tab-delimited text files include barcodes,gene names, and UMI counts for each Sample.
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Submission date |
Mar 15, 2022 |
Last update date |
Jul 31, 2022 |
Contact name |
Vittorio Sartorelli |
Organization name |
NIH
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Department |
NIAMS
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Street address |
50 South Dr.
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City |
Bethsda |
State/province |
MD |
ZIP/Postal code |
20814 |
Country |
USA |
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Platform ID |
GPL24247 |
Series (1) |
GSE198730 |
Transcriptomics, Regulatory Syntax, and Enhancer Identification in Heterogenous Populations of Mesoderm-Induced ESCs at Single-Cell Resolution |
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Relations |
BioSample |
SAMN26686107 |
SRA |
SRX14498752 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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