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Sample GSM2308736

Query DataSets for GSM2308736

Status

Public on Sep 26, 2017

Title

P50_rEC_rep1

Sample type

SRA

Source name

retinal endothelial cells

Organism

Mus musculus

Characteristics

strain: C57BL/6
tissue: retinal endothelial cells
age: postnatal day 50

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
rEC_all_FPKMs.xlsx

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

E-mail(s)

zionjeong@gmail.com

Phone

+4917631115236

Organization name

Max-Planck-Institute for Molecular Biomedicine