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Sample GSM3762876 Query DataSets for GSM3762876
Status Public on Apr 08, 2020
Title Dnttip1KO2_D1E6_rep2
Sample type SRA
Source name embryonic stem cells
Organism Mus musculus
Characteristics genotype: Dnttip1 KO2 (D1E6)
cell type: embryonic stem cells
Growth protocol mESCs were grown in chemically defined 2iL medium (defined naïve culture conditions): 50% DMEM/F-12; 50% Neurobasal Medium (no L-glutamine); B-27 Supplement, minus vitamin A (1:100); N-2 Supplement (1:200); 2 mM L-Glutamine or GlutaMAX (1:100); 0.1 mM β-Mercaptoethanol (1:500); 3 µM CHIR 99021 (GSK3β inhibitor) (1:1000 from 3 mM stock in DMSO); 1 µM PD 0325901 (MEK inhibitor) (1:1000 from 1 mM stock in DMSO); 1000 U/ml LIF (1:10,000 from 107 U/ml stock); 50 U/ml Pen/Strep (1:100).
Extracted molecule total RNA
Extraction protocol RNA was isolated from 3 x 10e6 cells with the RNeasy Mini Kit (Qiagen, 74106) following the manufacturer’s instructions with the following alterations. Cells were resuspended in 600 μl RLT buffer (with 2-Mercaptoethanol) and the homogenate further passed through a QiaShredder column (Qiagen, 79656) by centrifugation in a table top centrifuge for 2 minutes at full speed at room temperature (RT). The optional step after the second wash with RPE buffer was applied to dry the membrane. RNA was eluted with 85 μl H2O and supplied with 10 μl 10x DNAase buffer and 5 μl DNAse I (NEB, M0303S), mixed and incubated at RT for 20 min. After incubation RNA was purified by following the “RNA Cleanup” protocol in the RNeasy Mini Handbook. The optional step after the second wash with RPE buffer was applied to dry the membrane. RNA was eluted in 50 μl H2O and concentration determined with a NanoDrop 8000 Spectrophotometer.
RNA-seq Library Preparation and Sequencing:RNA was quantified using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific, R11490) and quality checked with the RNA 6000 Nano Kit (Agilent, 5067-1511) on a 2100 Bioanalyzer (Agilent, G2939BA) or High Sensitivity RNA ScreenTape Assay (Agilent, 5067-5579, 5067-5580, 5067-5581) on a 4200 TapeStation (Agilent, G2991AA) prior to library generation. Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina, 20020599) according to the manufacturer’s instructions. Libraries were analyzed for insert size distribution with the High Sensitivity DNA Kit (Agilent, 5067-4626) on a 2100 Bioanalyzer or the High Sensitivity D1000 ScreenTape Assay (Agilent, 5067-5584, 5067-5585, 5067-5587, 5067-5603) on a 4200 TapeStation. Libraries were quantified using the Quant-iT PicoGreen dsDNA Assay (Thermo Fisher Scientific, P11496). Paired end 100 cycle sequencing was performed on a HiSeq 2500, HiSeq 4000, or NovaSeq 6000 System (all from Illumina) according to the manufacturer’s instructions.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Data processing Total stranded RNA sequencing data were generated and mapped against mouse genome assembly NCBIM37.67 using the StrongArm pipeline described previously (Wu et al., 2016). Gene level quantification values were obtained with HT-seq based on the GENCODE annotation (vM20) and normalized by the TMM method with edgeR (version 3.16.5). Differential expression analysis was performed with the voom method applying the limma pipeline in R (version 3.3.1). Significantly up- and down- regulated genes were defined by at least a 1.5 fold change in gene expression and a p-value < 0.01. Reactome and gene set enrichment analysis (GSEA) were carried out using GSEA or EnrichR, respectively. Gene expression log2 CPM (counts per million) values were computed for heatmap and box plot visualization. Log2 FPKM gene expression values were applied for bar plot diagrams.
Genome_build: mm9
Submission date May 12, 2019
Last update date Apr 08, 2020
Contact name Hans-Martin Herz
Phone 901-595-2058
Organization name St. Jude Children's Research Hospital
Street address 262 Danny Thomas Place
City Memphis
State/province TN
ZIP/Postal code 38105-3678
Country USA
Platform ID GPL24247
Series (2)
GSE131060 The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression to control neurite outgrowth [RNA-Seq]
GSE131062 The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression to control neurite outgrowth
BioSample SAMN11632852
SRA SRX5822695

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

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