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Sample GSM4159468 Query DataSets for GSM4159468
Status Public on Jul 01, 2020
Title ESC_SL_GRO-seq_rep1
Sample type SRA
 
Source name mouse embryonic stem cells
Organism Mus musculus
Characteristics cell type: mouse embryonic stem cells
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.
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.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
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”.
 
Submission date Nov 13, 2019
Last update date Jul 02, 2020
Contact name Yiwei Lai
E-mail(s) lai_yiwei@gibh.ac.cn
Organization name Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences
Department South China Institute for Stem Cell Biology and Regenerative Medicine Key Laboratory of Regenerative Biology, CAS
Lab Miguel
Street address 190 Kai Yuan Avenue, Science Park
City Guangzhou
State/province Guangdong
ZIP/Postal code 510530
Country China
 
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]
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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