|
| Status |
Public on Oct 13, 2020 |
| Title |
5_RNAseq_mESC_serum_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
E14 mouse embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
modification: 3xFlag knock-in to endogeous Kdm2b C-terminus
genotype: wild-type
strain: 129P2/OlaHsd
|
| Growth protocol |
The wild-type E14 mESCs with 3xFlag knock-in to the endogenous Kdm2b gene were maintained on the 0.1% gelatin coated plates in "2i" medium that contained 50% Neurobasal medium (Life Technologies) and 50% DMEM/F12 medium (Life Technologies) supplemented with 1x N2-supplement (Life Technologies), 1x B27 supplement (Life Technologies), 7.5% bovine serum albumin (Life Technologies), 1x GlutaMAX (Life Technologies), 1x beta-mercaptoethanol (Life Technologies), 1mM PD0325901, 3mM CHIR99021, 1000 units/ml leukemia inhibitory factor (ESGRO, EMD Millipore), and 100U/ml penicillin/streptomycin (Life Technologies). The PD culture conditoin is the same as 2i culture condtion except for it containng only 1mM PD0325901. The serum-containing mESC culture condition included DMEM medium (Life Technologies) supplemented with 100U/ml penicillin/streptomycin (Life Technologies), 15% fetal bovine serum (Sigma), 1x nonessential amino acid, 1x sodium pyruvate (Life Technologies), 1x GlutaMAX (Life Technologies), 1x beta-mercaptoethanol (Life Technologies) and 1000 units/ml leukemia inhibitory factor (ESGRO, EMD Millipore).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted and purified from cells using QI shredder (Qiagen) and RNeasy (Qiagen) spin columns.
Total RNA (1 µg) was used to generate RNA-seq library using NEBNext Ultra Directional RNA library Prep Kit for Illumina (New England BioLabs, Inc) according to the manufacturer’s instructions. Adapter-ligated cDNA was amplified by PCR and followed by size selection using agarose gel electrophoresis. The DNA was purified using Qiaquick gel extraction kit (Qiagen) and quantified both with an Agilent Bioanalyzer and Invitrogen Qubit. The libraries were diluted to a working concentration of 10nM prior to sequencing. Sequencing on an Illumina HiSeq4000 instrument was carried out by the Genomics Core Facility at Michigan State University.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
serum medium
1_cuffdiffout_mESC_serum_2i_PD_gene_exp.diff
|
| Data processing |
For the RNA-Seq data analysis, all sequencing reads were mapped to NCBI build 37 (mm9) of the mouse genome using Tophat2.
The mapped reads were normalized to reads as Reads Per Kilobase of transcript per Million mapped reads (RPKM).
The differential gene expression was calculated by Cuffdiff program and the statistic cutoff for identification of differential gene expression is q < 0.05 and 1.5-fold RPKM change between samples.
Genome_build: mm9
|
| |
|
| Submission date |
Sep 09, 2020 |
| Last update date |
Oct 13, 2020 |
| Contact name |
Jin He |
| E-mail(s) |
hejin1@msu.edu
|
| Phone |
5173530613
|
| Organization name |
Michigan State Univeristy
|
| Street address |
603 Wilson Road, Biochem Bldg. Rm. 410
|
| City |
East Lansing |
| State/province |
Michigan |
| ZIP/Postal code |
48824 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (2) |
| GSE157747 |
Cell signaling coordinates global Polycomb Repressive Complex 2 recruitment and developmental gene expression in murine embryonic stem cells (RNA-Seq) |
| GSE157749 |
Cell signaling coordinates global Polycomb Repressive Complex 2 recruitment and developmental gene expression in murine embryonic stem cells |
|
| Relations |
| BioSample |
SAMN16090757 |
| SRA |
SRX9101809 |
