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Status |
Public on Mar 06, 2024 |
Title |
E7.5_rep3 |
Sample type |
SRA |
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Source name |
Embryo
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Organism |
Mus musculus |
Characteristics |
tissue: Embryo cell line: Strain: CD-1; embryo sex: male/female genotype: wild-type treatment: no treatment
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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)
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
E7.5_Epi_rep3 RNA_TPM.tsv
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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>)
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Submission date |
Sep 14, 2022 |
Last update date |
Mar 06, 2024 |
Contact name |
Helene Kretzmer |
E-mail(s) |
kretzmer@molgen.mpg.de
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Organization name |
Max Planck Institute for Molecular Genetics
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Department |
Genome Regulation
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Lab |
Meissner Lab
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Street address |
Ihnestraße 63
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City |
Berlin |
ZIP/Postal code |
14195 |
Country |
Germany |
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Platform ID |
GPL24247 |
Series (1) |
GSE213336 |
Basal cell extrusion primes pluripotent cells for differentiation |
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Relations |
BioSample |
SAMN30852862 |
SRA |
SRX17578097 |
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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