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| Status |
Public on Sep 27, 2022 |
| Title |
RNA-Klf5_KD-3 |
| Sample type |
SRA |
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| Source name |
mouse trophoblast stem cells
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| Organism |
Mus musculus |
| Characteristics |
genotype: Klf5-KD TSCs.
cell type: mouse trophoblast stem cells
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| Growth protocol |
Trophoblast stem cells were cultured in 70% feeder condition medium + 30% TS medium (RPMI1640 + 20% FBS + 0.1M 2-mercaptoethanol + 2 mM glutamine + 1 mM sodium pyruvate + 100× Pen/Strep) + 25 ng/ml Fgf4 (Peprotech) + 1 μg/ml heparin (Sigma) on mitomycin C treated mouse embryonic fibroblasts (MEFs). For partially differentiated medium, we removed Fgf4 from TSCs maintained culture medium.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA-seq: Total RNA was extracted from cells by Trizol (Invitrogen, 15596-018), and 1 μg total RNA was reverse transcribed to obtain cDNA with cDNA Synthesis Kit (Vazyme, R212-01) according to the manufacturer’s protocol. ChIP-seq: For ChIP-seq, TSCs were crosslinked with 1% formaldehyde for 10 min, and quenched by 2 mL of 125 mM glycine for 5 min rotation at room temperature. Cells were washed 3 times with cold PBS. After centrifugation, cell pellets were lysed in buffer (50 mM HEPES-KOH, 140 mM NaCl, 1 mM EDTA, pH 8.0, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 50 mM Tris-HCl, pH 8.0) for 10 min at 4℃. Chromatin was sonicated to 100-500 bp for the subsequent experiments. ATAC-seq: ATAC-seq was performed as previously report[50]. In brief, a total of 50,000 cells were washed once with 50 µl of cold PBS and resuspended in 50 µl lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). After centrifugation at 4℃, suspension of nuclei diluted to 5 ng/µl in the elution buffer (10 mM Tris buffer, pH 8) and followed by the addition of 50 µl transposition reaction mix of DNA Library Prep Kit V2 for Illumina (Vazyme, TD501).
RNA-seq: Total RNA was extracted from TSCs by Trizol and RNA-Seq library were generated using RNA Library Prep Kit for Illumina® (NEB, E7530L) according to the manufacturer’s recommendations. ChIP-seq: The samples were pre-incubated with protein A/G Dynabeads (Life Technologies, 10015D) and the beads were then removed. Subsequently, the sample was added with 8 mg of Klf5(Sigma, 09-822) or H3K27ac antibody and rotated overnight at 4℃. Beads were washed four times with a Wash buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% glycerol, 0.5% Triton X-100, 0.01% SDS), eluted and reverse crosslinked. Paired-end 125 bp sequencing was performed on Illumina Hiseq X-Ten. ATAC-seq: The reaction mix was gently mixed and incubated at 55℃ for 10 min. ATAC-seq library was amplified in 9 cycles. Libraries were purified using Qiagen PCR Cleanup Kit according to the manufacturer’s instructions. Library concentrations were measured by KAPA Library Quantification Kit (KAPA Biosystems, KK4824) according to the manufacturer's instructions and checked by gel electrophoresis. Lastly, ATAC libraries were sequenced on Illumina Hiseq X-Ten.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Data processing |
RNA-seq: Reads of RNA-seq were mapped to the mm10 genome with STAR[51] using the ENCODE standard options. Gene expression levels were quantified by Stringtie using the refFlat database[52] from the UCSC genome browser. Differentially expressed genes follow this criteria: fold change > 2, FDR < 0.05 in knockdown sample for upregulated genes, fold change < 0.5, FDR < 0.05 and in control sample for downregulated genes.GO enrichment analyses of differentially expressed genes was used in DAVID. ChIP-seq: Reads were trimmed using TrimGlare and then mapped to the mm10 genome using Bowtie2 (v4.4.7) with parameters '-X 2000'. Multi-mapped and unmapped and low-quality reads were removed using samtools (v1.11) (10.1093/bioinformatics/btp352) and PCR duplicates were removed using the MarkDuplicates command from Picard tools (v2.6.0). ChIP-Seq peaks were called using MACS2 (v2.2.6) (10.1186/gb-2008-9-9-r137) with '-q 0.01' parameters. Annotation was performed using ChIPseeker version 1.22.1 (10.1093/bioinformatics/btv145). ATAC-seq: Raw files (fastq format) processed through the ATAC-seq pipeline published by ENCODE. Briefly, reads were trimmed, filtered, and aligned to mm10. All unmapped reads, reads with low mapping quality (MAPQ < 30) and PCR duplicates were removed. MACS2 was used to call peaks for each individual sample with the "-nomodel" and "--shift -100 --extsize 200" parameters. The bam files of the replicates were then merged together using Samtools. Peak files were merged using Bedops (v2.4.38) (10.1093/bioinformatics/bts277) merge to obtain a consensus set of peaks across samples. The number of reads that fell into the peaks was obtained using bedtools multicov (v2.27.0) (10.1093/bioinformatics/btq033). Then we normalized the read counts by computing the numbers of reads per kilobase of bin per million of reads sequenced (RPKM) by custom scripts. Limma was used for determining hyper and hypo-accessible peaks across different samples using default parameters. Peaks were called as hyper or hypo-accessible using abs( log2 fold change ) > 0.5 and adjusted P < 0.05. For each sample, the Pearson correlation between replicates was calculated using the peaks that had an RPKM > 5 in at least one of the replicates.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample
Supplementary files format and content: bigWig(except for Input sample)
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| Submission date |
Jun 27, 2022 |
| Last update date |
Sep 27, 2022 |
| Contact name |
Wu Linhui |
| E-mail(s) |
[email protected]
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| Phone |
18627104808
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| Organization name |
Huazhong Agricultural University
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| Department |
College of Animal Science and Veterinary Medicine
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| Lab |
Miao Lab
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| Street address |
No.1 Shizishan Street
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| City |
Wuhan |
| State/province |
Hubei |
| ZIP/Postal code |
430070 |
| Country |
China |
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| Platform ID |
GPL21273 |
| Series (1) |
| GSE207053 |
Klf5 regulates trophoblast stem cell pluripotency as a transcription activator via H3K27ac dependent chromatin accessibility |
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| Relations |
| BioSample |
SAMN29381200 |
| SRA |
SRX15922026 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6276320_RNA-Klf5_KD-3.counts.txt.gz |
3.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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