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Sample GSM5242303 Query DataSets for GSM5242303
Status Public on Apr 15, 2024
Title shPdgf-d mESCs-1
Sample type SRA
 
Source name shPdgf-d mESCs
Organism Mus musculus
Characteristics cell line: E14
cell type: embryonic stem cells
passage: passage 25
treatment: transfected with pLent-Pdgf-d-4in1-shRNA plasmids
Treatment protocol To generate stable Pdgf-d knockdown mESCs, 1μg pLent-Pdgf-d-4in1-shRNA plasmids were transfected into HEK293T cells together with 2μg lentivirus packing plasmids pSPAX2 and pMD2G using Lipofectamine 2000. Supernatants containing the viral particles was collected and added to the mESCs. New ESC medium was added 6 hours later. Cells were selected by puromycin (2 μg/ml). pLent-4in1-shRNA-GFP-Puro was used as a negative control.
Growth protocol Mouse embryonic stem cells (mESCs) were maintained in culture dish coated with 0.1% gelatin in ESC medium, which contained Knockout DMEM medium and supplemented with 15% (v/v) fetal bovine serum , β–mercaptoethanol (0.1µM), GlutaMax-I supplement (2mM), MEM non-essential amino acids (0.1 mM), 1% (v/v) Penicillin-Streptomycin, LIF (1,000 U/ml), and 2 inhibitors (CHIR99021, PD325901 at 3 µM and 1 µM, respectively)
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from shControl or shPdgf-d mESCs by Trizol regent. 1 ∼ 2 µg of total RNA was used for the construction of sequencing libraries by KAPA Stranded RNA-Seq Library Prep Kit(Illumina).
RNA libraries for sequencing were prepared following standard Illumina protocols
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Data processing Solexa pipeline version 1.8 (Off-Line Base Caller software, version 1.8) used for basecalling.
Sequenced reads were trimmed for adaptor sequence by fastQC, and masked for low-complexity or low-quality sequence, then mapped to GRCm38 mouse genome using Hisat2 software.
Fragments per kilobase of transcript per million mapped reads (FPKM) were calculated using StringTie and R software Ballgown package.
Genome_build: GRCm38
Supplementary_files_format_and_content: An Excel file include FPKM values for all Samples
 
Submission date Apr 15, 2021
Last update date Apr 15, 2024
Contact name Weisi Lu
E-mail(s) luweisi3@mail.sysu.edu.cn
Organization name Zhongshan Ophthalmic Center, Sun Yat-sen University
Department State Key Laboratory of Ophthalmology
Lab Xuri Li Lab
Street address No.7 Jinsui Road
City Guangzhou
ZIP/Postal code 510060
Country China
 
Platform ID GPL24247
Series (1)
GSE172117 RNA sequencing for the gene expression profile changes of shPdgf-d and shControl mESCs
Relations
BioSample SAMN18746702
SRA SRX10605638

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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