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| Status |
Public on Jul 01, 2020 |
| Title |
ESC_SL_GRO-seq_rep1 |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cells
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| Growth protocol |
ESCs in SL medium were cultured in DMEM/high glucose containing 15% FBS, GlutaMax, nonessential amino acids, sodium pyruvate, β-mercaptoethanol, and 1,000 U/ml LIF on mitomycin-C treated mouse embryonic fibroblasts (MEFs), and they were split onto 0.2% gelatin pre-coated plates prior to the experiment. ESCs in 2iL medium were cultured in a 1:1 mix of DMEM/F12 and Neurobasal medium, with N2 and B27 supplements, GlutaMax, nonessential amino acids, sodium pyruvate, β-mercaptoethanol, 1,000 U/ml LIF, 3 μM, and 1 μM PD0325901 on 0.2% gelatin pre-coated plates.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Nuclei from ESCs were extracted, and run-on-transcribed with BrUTP (Sigma, B7166) and other NTPs at 30 °C for 5 min. Nascent RNA was enriched by agarose coated anti-BrUTP (Sant Cruz, sc-32323). Poly(A) tail was added to the nascent RNA by Poly(A) Polymerase (NEB, M0276S) to synthese cDNA with oligo(dT) primer. GRO-seq libraries were amplified by PCR for 10 cycles and seperated by 10% TBE gel. The bands ranging from 160 bp to 300 bp were cutted and purified by isopropanol precipitation.
Libraries were sequenced on a NovaSeq 6000 sequencer according to the manufacturer’s instructions with 12Gbase PE150 reads.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: GRO-seq
Illumina bcl2fastq2.17 software was used for base-calling.
For GRO-seq, adaptors were first trimmed with fastp (v0.20.0), only read1 were kept for further analysis. PCR duplicates were collapse using Fastx-toolkit. 20 bp PolyA sequence and 8 bp random sequence were trimmed from 3’end.
Cleandata were then aligned to the mm10 mouse genome assembly using Bowtie2.
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig files were generated using bamCoverage script of deepTools with the options “ --binSize 10 --normalizeUsingRPKM”.
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| Submission date |
Nov 13, 2019 |
| Last update date |
Jul 02, 2020 |
| Contact name |
Yiwei Lai |
| E-mail(s) |
[email protected]
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| Organization name |
Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences
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| Department |
South China Institute for Stem Cell Biology and Regenerative Medicine Key Laboratory of Regenerative Biology, CAS
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| Lab |
Miguel
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| Street address |
190 Kai Yuan Avenue, Science Park
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| City |
Guangzhou |
| State/province |
Guangdong |
| ZIP/Postal code |
510530 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE123692 |
β-catenin safeguards the ground state of pluripotency by strengthening the robustness of the transcriptional apparatus |
| GSE140340 |
β-catenin safeguards the ground state of pluripotency by strengthening the robustness of the transcriptional apparatus [GRO-seq] |
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| Relations |
| BioSample |
SAMN13279906 |
| SRA |
SRX7135447 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4159468_ESC_SL_GRO-seq_rep1.bw |
33.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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