title: Illumina MiSeq paired end sequencing; Actively translating transcriptome (Ribotag) profiling of mouse retinal endothelial cells during postnatal development
design: Actively translating transcriptome (Ribotag) profiling of mouse retinal endothelial cells during postnatal development
library_construction_protocol: HA-tagged Mafb overexpressed MS1 ECs were cross-linked with 1% (wt/vol) formaldehyde for 10 minutes at room temperature and then added glycine to a final concentration of 0.15M. Chromatin was sonicated to an average fragment length of 150-250 bp using SFX250 digital sonifier (Branson) for 2 minutes: 0.7 second on / 1.3 second off on ice and immunoprecipitated with anti-HA antibody-conjugated magnetic beads (M180-11; MBL) overnight at 4°C. Following protein-DNA complex elution, samples were treated with RNaseA and proteinase K and incubated at 65°C overnight for reverse cross-linking. Immunoprecipitated DNA was purified using AMPure XP SPRI beads (Beckman Coulter).  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.  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.  Sequencing libraries were prepared with NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs)