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Sample GSM4797288 Query DataSets for GSM4797288
Status Public on Aug 31, 2021
Title KO.2iL
Sample type SRA
Source name 2iL
Organism Mus musculus
Characteristics genotypes: Dnmt3a3b double KO
cell line: 2iL
Treatment protocol After the harvest, live cells were collected by FACS moflo.
Growth protocol Mouse ES cells were cultured in 2iL. EpiLC differentiation were performed in actinvin a, bFgf and 1% KSR medium.
Extracted molecule total RNA
Extraction protocol 10xgenomics
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Data processing Preliminary sequencing results (bcl files) were converted to fastq files with CellRanger (version 3.0) following the standard 10x Genomics protocol. Barcodes and unique molecular identifier (UMI) ends were trimmed to 26 bp, and mRNA ends to 98 bp.
Reads were then aligned to the mouse reference genome (mm10) and gene counts were obtained using the GRCm38.92 annotation in CellRanger.
We used Seurat (version 2.3.0) (Butler et al., 2018) to further process the single-cell RNA-seq data. Cells that have between 200 and 5500 unique gene counts, and with less than 5% mitochondrial gene content were filtered
Log-normalization using a scale factor of 10,000 was performed, and the data was scaled to produce standardized expression values for each gene across all cells (z-score transformation), while also regressing out unwanted variation in the percent of mitochondrial gene content.
We determined the top 2,000 most highly variable genes, and performed principal component analysis. Clusters were determined using the Louvain community detection method as implemented by the FindCluster function in Seurat with a resolution of 0.9. Visualisation was performed after using tSNE dimensional reduction on the first 18 principal components. Differential expression was performed using the Wilcoxon rank sum test, using a threshold of log2 fold change greater than 0.1 and p-value less than 0.05.
Genome_build: mm10
Supplementary_files_format_and_content: Gene expression matrix and cell type identities
Submission date Sep 21, 2020
Last update date Aug 31, 2021
Contact name Irina Mohorianu
Organization name University of Cambridge
Department Wellcome-MRC Cambridge Stem Cell Institute
Street address Puddicombe Way
City Cambridge
ZIP/Postal code CB2 0AW
Country United Kingdom
Platform ID GPL13112
Series (2)
GSE158310 De novo DNA methylation suppresses aberrant fate trajectory during epiblast transition [RNA-seq]
GSE158347 Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory
BioSample SAMN16233423
SRA SRX9163332

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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