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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 02, 2023 |
| Title |
RNAseq_TSC1_KDM2BKO_Rep2 |
| Sample type |
SRA |
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| Source name |
Trophoblast stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Trophoblast stem cells
condition: KDM2B KO
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| Treatment protocol |
On the day of collection, TSCs were rinsed once with DPBS (Thermo, #14190144) and detached using Trypsin-EDTA (0.05%)(Thermo, #25300054). The cells were spun down for 4 min at 1000 rpm, counted and flash frozen on dry ice. The cell pellets were stored at -80°C.
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| Growth protocol |
Trophoblast stem cells (TSCs) cells were grown on MEF-coated cell culture dishes containing TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149). Splitting was carried out every five to seven days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using Trypsin-EDTA (0.05%)(Thermo, #25300054). TSCs were passaged in clumps. Before sample collection, TSCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in MEF-conditioned TSC medium (70 % MEF-conditioned medium, 30 % TSC medium, +FGF4 (37.5 ng/ml), Heparin (1.5 µg/ml)).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cell pellets were resuspended in 350 µl RLT Plus buffer containing 1% 2-mercaptoethanol (Thermo, #21985023). After cell lysis by trituration and vortexing, RNA was extracted using RNeasy Plus Micro Kit (Qiagen, #74034) and RNA concentration and quality was measured using the Agilent RNA Screen Tape (Agilent Technologies, #5067- 5576) on an Agilent 4150 Tapestation system. All samples analyzed had a RINe value higher than 8,0, and were subsequently used for library preparation.
mRNA libraries were prepared using KAPA Stranded RNA-Seq Kit (KapaBiosystem, #KK8421/07962207001) according to the manufacturer’s instructions. 500 ng of total RNA was used for each sample to enter the library preparation protocol. For adapter ligation dual indexes were used (NEXTFLEX® Unique Dual Index Barcodes #NOVA-514150 and #NOVA-514151) at a working concentration of 71 nM (5 µl of 1 µM stock in each 70 µl ligation reaction). Twelve library PCR cycles were used. Quality and concentration of the obtained libraries were measured using Agilent High Sensitivity D5000 ScreenTape (Agilent-Technologies, #5067- 5592) on an Agilent 4150 TapeStation.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
RNA sequencing of TSCs
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| Data processing |
Raw reads were subjected to adapter and quality trimming with cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25 --interleaved --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC), followed by poly-A trimming with cutadapt (parameters: --interleaved --overlap 20 --minimum-length --adapter "A[100]" --adapter "T[100]").
Reads were aligned to the mouse reference genome (mm10) using STAR (version 2.7.5a; parameters: --runMode alignReads --chimSegmentMin 20 --outSAMstrandField intronMotif --quantMode GeneCounts) and transcripts were quantified using stringtie (version 2.0.6; parameters: -e) with GENCODE annotation (release VM19).
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited file containing gene abundances: <Gene ID> <Gene Name> <Reference> <Strand> <Start> <End> <Coverage> <FPKM> <TPM>
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| Submission date |
Mar 07, 2022 |
| Last update date |
Jan 02, 2023 |
| Contact name |
Sara Hetzel |
| E-mail(s) |
hetzel@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 (2) |
| GSE166362 |
Dynamic antagonism between key repressive pathways maintains the placental epigenome |
| GSE198076 |
Dynamic antagonism between key repressive pathways maintains the placental epigenome (RNA-seq) |
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| Relations |
| BioSample |
SAMN26510724 |
| SRA |
SRX14401519 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5938152_RNAseq_TSC1_KDM2BKO_Rep2_mm10_stringtie.tsv.gz |
1.3 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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