title: NextSeq 500 sequencing; RNA-Seq Analysis of mouse endothelial cells from multiple organs
design: RNA-Seq Analysis of mouse endothelial cells from multiple organs
library_name: heart4_s
library_construction_protocol: We isolated endothelial cells (EC) from various FA-handling organs using the RiboTag method, which precisely tags the ribosomal subunit with hemagglutinin (HA) in VE-Cadherin expressing cells. RiboTag mouse was crossed with VE-Cadherin cre mice to isolate polysome-bound mRNAs of VE-Cadherin expressing cells (EC). Indicated tissues were harvested and immediately snap frozen. Then, polysome buffer (50 mM Tris, pH 7.5, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 200 U/ml RNasin, 1 mg/ml heparin, 100 μg/ml cyclohexamide, and 1× protease inhibitor mixture) were added to each sample and homogenized using Precellys lysis kit (Bertin). For immunoprecipitation (IP) against HA, anti-HA antibody-conjugated magnetic beads (M180-11; MBL) were added to the supernatant after centrifugation for 10 min at 12000rpm 4 °C, and incubated on an rotating shaker at 4 °C overnight. Then, 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, 1 mM DTT, and 100 μg/ml cyclohexamide) and resuspended in 500 μl of TRIzol® Reagent (Invitrogen). Total RNAs were extracted using the RNA isolation mentioned in methods. The quality and quantity of the RNA samples were analyzed using Agilent 2100 Bioanalyzer with RNA 6000 pico kit (Agilent), and further performed to RNA-seq. Total RNA was extracted from the samples using TRIzol® Reagent (Invitrogen) according to the manufacturer's instructions.  SENSE 3' mRNA-Seq Library Prep Kit (Lexogen, Inc.) were used according to the manufacturer's instructions. In brief, each 500ng total RNA were prepared and an oligo-dT primer containing an Illumina-compatible sequence at its 5' end was hybridized to the RNA and reverse transcription was performed. After degradation of the RNA template, second strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5' end. The double-stranded library was purified by using magnetic beads to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The finished library is purified from PCR components. High-throughput sequencing was performed as single-end 75 sequencing using NextSeq 500 (Illumina, Inc.).
attributes.tag: Experimental Factor: organism part
attributes.value: heart
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: anti-HA (M180-11; MBL)