|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Mar 06, 2024 |
Title |
EpiSC_rep1 |
Sample type |
SRA |
|
|
Source name |
cell line
|
Organism |
Mus musculus |
Characteristics |
tissue: cell line cell line: 2D epiblast stem cells cell type: Mouse pluripotent stem cells genotype: wild-type treatment: Conversion of ESCs via FGF2-ActivinA treatment
|
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 |
EpiSC_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
|
Lab |
Meissner Lab
|
Street address |
Ihnestraße 63
|
City |
Berlin |
ZIP/Postal code |
14195 |
Country |
Germany |
|
|
Platform ID |
GPL24247 |
Series (1) |
GSE213336 |
Basal cell extrusion primes pluripotent cells for differentiation |
|
Relations |
BioSample |
SAMN30852882 |
SRA |
SRX17578072 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
|
|
|
|
|