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Sample GSM6579138

Query DataSets for GSM6579138

Status

Public on Mar 06, 2024

Title

E6.5_rep2

Sample type

SRA

Source name

Embryo

Organism

Mus musculus

Characteristics

tissue: Embryo
cell line: Strain: CD-1; embryo sex: male/female
genotype: wild-type
treatment: no treatment

Extracted molecule

total RNA

Extraction protocol

RNA was extracted from the epiblast of E3.5, E4.5, E5.5, E6.5, and E7.5 following the manufacturer's protocol (PicoPure RNA isolation kit; KIT0204; ThermoFisher Scientific) and eluted in 7 L elution buffer. The full eluate was utilized to prepare the library. 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 SMARTer Stranded Total RNA-Seq Kit v3 – Pico Input kit (634485, Takara Bio) following the manufacturer's instructions, with the exceptions listed below. To account for the input differences, fragmentation of RNA was done for 6 minutes at 85 °C on E3.5 and E4.5 epiblast samples, and for 4 minutes at 94 °C on E5.5, E6.5, and E7.5 epiblast samples. Adapters and indices from Illumina were added to distinguish the libraries (SMARTer RNA Unique Dual Index kit, 634451, Takara Bio). Instead of NucleoMag beads, the libraries were purified with AMPure XP beads (A63880, Beckman Coulter). ZapR v3 and R-probes v3 were used to deplete ribosomal cDNA (supplied with the kit). Libraries were amplified for thirteen cycles, purified, and analyzed for quality and concentration 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

E6.5_Epi_rep2
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