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Sample GSM2308740 Query DataSets for GSM2308740
Status Public on Sep 26, 2017
Title P6-MafbcKO_rEC_rep2
Sample type SRA
Source name retinal endothelial cells
Organism Mus musculus
Characteristics strain: C57BL/6
tissue: retinal endothelial cells
age: postnatal day 6
Treatment protocol (Sample 19/20) HA-tagged Mafb construct was amplified by PCR, sub-cloned into pCR8/GW/TOPO (Invitrogen), and cloned with pLEX_307 (Addgene, #41392) by an LR recombination reaction. To produce lentiviruses, HEK293 cells were plated at a density of 8 x 106 per 10 cm dish. On the next day, the cells were transfected with 3μg of psPAX2 (Addgene, #12260), 1.5μg of pMD2.G (Addgene, #12259) and 4.5μg of lentiviral vector pLEX_307-HA-Mafb using 27μl Fugene 6 in 600µl Opti-MEM per dish. Virus-containing supernatants were collected at 24 hour, filtered through a 0.4-µm PVDF filter (Millipore), concentrated at 20,000 rpm for 2 hours using a optima L-100 XP ultracentrifuge (Beckman Coulter), re-suspended in knockout DMEM and then stored at -80°C until use. The lentivirus-infected cells were then subcultured and selected in puromycin (Sigma) or blasticidin (Calbiochem) to generate stable cell lines.
Growth protocol (Sample 19/20) MS1 mouse pancreas ECs (CRL-2279; ATCC) were cultured in DMEM medium supplemented with 5% fetal calf serum (FCF) at 37°C and 5% CO2.
Extracted molecule total RNA
Extraction protocol Mouse retinas were isolated, immersed in liquid nitrogen immediately to snap freeze, and stored at -80 °C for later use. We added 400μl of polysome buffer (50 mM Tris, pH 7.5, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1mM DTT, 200 U/ml RNasin, 1 mg/ml heparin, 100 μg/ml cyclohexamide, and 1x protease inhibitor mixture) to each sample and disrupted the tissue in 1.5 ml microcentrifuge tubes using pellet pestles (749540-0000; Kimble Chase). Postmitochondrial supernatant was formed by centrifugation for 10 minutes at 12,000 rpm at 4 °C and 10μl of the supernatant were saved for input. For IP against HA, we added 25μl of anti-HA antibody-conjugated magnetic beads (M180-11; MBL) to the retinal supernatant prior to incubation on an orbital shaker at 4°C overnight. IP beads were washed for four times with high-salt buffer (50 mM Tris, pH 7.5, 300 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1mM DTT, and 100 μg/ml cyclohexamide) and resuspended in 350μl of RLT plus buffer plus β-mercaptoethanol. Total RNAs were extracted using RNeasy Micro kit (Qiagen) according to manufacturer’s instructions.
RNA library construction was performed with the Illumina TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero (Illumina) according to the manufacturer’s instructions.
RNA-Seq / (Sample 19/20) ChIP-Seq
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina MiSeq
Description PdgfbiCre-Ribotag
Data processing Sequenced reads were aligned to the mouse (mm10) reference genome with TopHat (version 2.0.12)
The aligned reads were used to quantify mRNA expression by using HTSeq-count (version 0.6.1)
DESeq2 (Love et al., 2014) was used to identify DEGs across the samples.
Genome_build: mm10
Supplementary_files_format_and_content: Xlsx file include FPKM values for each sample / (Sample 19/20) tab-delimited text file include peaks called by Bowtie2 followed by HOMER analysis.
Submission date Sep 09, 2016
Last update date May 15, 2019
Contact name Hyun-Woo Jeong
Phone +4917631115236
Organization name Max-Planck-Institute for Molecular Biomedicine
Department Dept. Tissue Morphogenesis
Lab Adams
Street address Roentgenstr.20
City Muenster
State/province NRW
ZIP/Postal code 48149
Country Germany
Platform ID GPL16417
Series (1)
GSE86788 Actively translating transcriptome (Ribotag) profiling of mouse retinal endothelial cells during postnatal development
BioSample SAMN05761468
SRA SRX2155773

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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