|
Status |
Public on Jan 01, 2016 |
Title |
MutantRescue_E294K_F10_RNAseq |
Sample type |
SRA |
|
|
Source name |
embryonic stem cells
|
Organism |
Mus musculus |
Characteristics |
strain: Stella:GFP genotype: Prdm14-/- + FLAG-HA-Prdm14 E294K mutant
|
Growth protocol |
For maintenance all mouse ESC lines were grown in so-called “2i+LIF” medium that is serum free N2B27-based medium supplemented with MEK inhibitor PD0325901 (0.8 μM) and GSK3β inhibitor CHIR99021 (3.3 μM) in tissue culture (TC) dishes pretreated with 7.5 μg/ml polyL-ornithine (Sigma) and 5 μg/ml laminine (BD) (Hayashi and Saitou, 2013; Hayashi et al., 2011). For ChIP-seq, RNA-seq and immunoprecipitation experiments Stella:GFP mESC lines and FH-Prdm14 derivatives were cultured for 5 days in feeder-free conditions in DMEM-high glucose medium (DMEM/high glucose; HyClone) containing 15% (v/v) FBS, 1 mM glutamine, penicillin/streptomycin, 0.1mM nonessential amino acids, 0.1 mM 2-mercaptoethanol and supplemented with LIF (serum+LIF conditions).
|
Extracted molecule |
total RNA |
Extraction protocol |
ChIPs were performed using approximately 1-2 x 10^7 mESCs fixed with 1% formaldehyde for 10 min at room temp. RNA was extracted using Trizol. Sequencing libraries were prepared starting from ~30ng of ChIP DNA or oligo-dT purified mRNA using the NEBNext Multiplex Oligos for Illumina kit (Cat# E7335S).
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|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
|
|
Data processing |
Reads aligned to mm9 using default settings with Bowtie2.2.4 Wig files were generated with QuEST2.4 RNA-seq were aligned to mm9 and quantified against mouse ENSEMBL 78 (GRCm38) gene models using htseq-count. Variance-stabilizing transformation (VST) was applied across samples for processed data. Genome_build: Alignments done using mm9 as reference Supplementary_files_format_and_content: wig and tab delimited text
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|
|
Submission date |
Oct 30, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Natalie Nady |
Organization name |
Stanford
|
Department |
Chemical and Systems Biology
|
Lab |
Wysocka
|
Street address |
265 Campus Dr.
|
City |
Stanford |
State/province |
CA |
ZIP/Postal code |
94303 |
Country |
USA |
|
|
Platform ID |
GPL19057 |
Series (1) |
GSE74547 |
ETO Family Protein Mtgr1 Mediates Prdm14 Functions in Stem Cell Maintenance |
|
Relations |
BioSample |
SAMN04227585 |
SRA |
SRX1404252 |