|
| Status |
Public on Jun 13, 2017 |
| Title |
Pcgf1KO+ Pcgf1_flag RNAseq |
| Sample type |
SRA |
| |
|
| Source name |
Pcgf1KO+ Pcgf1_flag, mESCs
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: mouse embryonic stem cells
genotype: Pcgf1KO+ Pcgf1_flag
|
| Treatment protocol |
Pcgf1 KO ES cells were generated by Cas9 technology. Pcgf1KO+Pcgf1_flag ES cells were established by lentiviral infection technology.
|
| Growth protocol |
ES cells were cultured with mouse embryonic fibroblasts (MEFs) inactivated with mitomycin-C in DMEM supplemented with15% fetal calf serum , leukemia inhibitory factor , L-glutamine , non-essential amino acids , 0.1 mM betamercaptoethanol , and penicillin/streptomycin. All ES cell lines were maintained at 37°C in 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the TRIzol reagent following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer.
A total amount of 2 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and RNase H. Second strand cDNA synthesis was subsequently performed using buffer, dNTPs, DNA polymerase I and RNase H. The library fragments were purified with QiaQuick PCR kits and elution with EB buffer, then terminal repair、A-tailing and adapter added were implemented. The aimed products were retrieved by agarose gel electrophoresis and PCR was performed, then the library was completed.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Mapping reads with the reference genome use TopHat 2.0.12
Assemble reads use Cufflinks 2.2.1
Gene expression level analysis HTSeq 0.6.0
Alternative splicing prediction use Cuffcompare 2.2.1
Differential expression gene analysis use DEGSeq 1.18.0
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample …
|
| |
|
| Submission date |
Feb 26, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Wukui Zhao |
| E-mail(s) |
zhaowukui163@163.com
|
| Phone |
86-02558641524
|
| Organization name |
Nanjing University
|
| Department |
Model Animal Research Center
|
| Lab |
MOE Key Laboratory of Model Animal for Disease Study
|
| Street address |
12 Xuefu Road
|
| City |
Nanjing |
| State/province |
Jiangsu |
| ZIP/Postal code |
210061 |
| Country |
China |
| |
|
| Platform ID |
GPL21273 |
| Series (1) |
| GSE95383 |
Expression regulation by Pcgf1 in mouse embryonic stem cells |
|
| Relations |
| BioSample |
SAMN06335400 |
| SRA |
SRX2577113 |
