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Sample GSM6579119

Query DataSets for GSM6579119

Status

Public on Mar 06, 2024

Title

ESC3D_rep1

Sample type

SRA

Source name

cell line

Organism

Mus musculus

Characteristics

tissue: cell line
cell line: 3D epiblast stem cells
cell type: Mouse pluripotent stem cells
genotype: wild-type
treatment: Cultured in 3D Matrigel with FGF2-ActivinA-XAV-Noggin

Extracted molecule

total RNA

Extraction protocol

RNA was isolated from the samples according to the manufacturer's instructions using the RNeasy kit (74004, Qiagen).
1 µg RNA was used as input for the library preparation. Prior to the construction of the library, the integrity and quality of the RNA were evaluated using a 4200 Agilent TapeStation device and RNA ScreenTape reagents (Agilent). Samples having a RIN > 7.0 for RNA integrity were used. The library was generated with the TruSeq Stranded mRNA capture kit (20020594, KAP Biosystems) following the manufacturer's instructions, with the exceptions listed below. Unique Dual-Indexed (UDI, KAPA biosystems) adapters were ligated and the libraries were amplified for 8 cycles. Quality and concentration were measured using a 4200 Agilent TapeStation instrument and D5000 ScreenTape reagents (Agilent).
Accel-NGS Methyl-Seq DNA Library Kit (manufacturer's recommendation)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

ESC_3D_rep1
RNA_TPM.tsv

Data processing

10X Genomic single-cell 3' RNA-seq
Raw base call (BCL) files generated by Illumina sequencers were demultiplexed into FASTQ files using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("mkfastq" pipeline, default parameters).
For each experiment FASTQ files were aligned, filtered, barcode and UMI counted using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("count pipeline", default parameters).
Assembly: mm10
Supplementary files format and content: For all samples there is one tar gz archive containing the gene-barcode matrices as produced by the Cell Ranger pipeline. They contain the MEX-formatted UMI count matrices of each sample, or if multiple libraries where sequence, the aggregated gene-barcode matrix.
PolyA RNA-seq
All RNAseq samples were pre-processed using cutadapt to remove adapter and trim low quality bases.
Reads were subsequently aligned against the human reference genome mm10 using STAR (parameter: outSAMtype BAM SortedByCoordinate --outSAMattributes Standard --outSAMstrandField intronMotif --outSAMunmapped Within --quantMode GeneCounts).
Stringtie was used for calculation of strand-specific TPMs.
Assembly: mm10
Supplementary files format and content: tsv of TPM values
Total RNA-seq
All RNAseq samples were pre-processed using cutadapt to remove adapter and trim low quality bases.
To remove rRNA all reads were first aligned against the rDNA databank (rfam-5s-database-id98) using STAR and unmapped reads were kept for the next step.
Reads were subsequently aligned against the human reference genome mm10 using STAR (parameter: outSAMtype BAM SortedByCoordinate --outSAMattributes Standard --outSAMstrandField intronMotif --outSAMunmapped Within --quantMode GeneCounts).
Stringtie was used for calculation of strand-specific TPMs.
Assembly: mm10
Supplementary files format and content: tsv of TPM values
Bisulfite-Seq
Bisulfite-converted reads were quality filtered and adapter trimmed using cutadapt (v2.4), followed by alignment to the reference genome with bsmap using the following parameter: -v 0.01 -s 16 -w 100 -S 1 -R -u -q 20. Duplicate reads were removed using GATK MarkDuplicates (v4.1.0.0).
Methylation rates were called using mcall using default parameter and subsequently filtered for coverage (min 10 reads, max 150 reads).
Assembly: mm10
Supplementary files format and content: bigWig file of 10X coverage filtered methylation rate BED file (<chr> <start> <end> <methylation rate>)

Submission date

Sep 14, 2022

Last update date

Mar 06, 2024

Contact name

Helene Kretzmer

E-mail(s)

kretzmer@molgen.mpg.de

Organization name

Max Planck Institute for Molecular Genetics

Department

Genome Regulation