Livers of WT (C57BL/6J) and Lyve-1-KO (B6.129S1-Lyve1tm1Lhua/J (JAX stock #006221)) were perfused with a 0.05 % collagenase containing amino acid/ saccharide calcium-deprived medium (C2674, Sigma Aldrich, St. Louis, MO, USA) via the portal vein. Mice were sacrificed and livers were excised. Livers were minced mechanically and digested at 38 °C in a collagenase/Gey’s balanced salt solution (G9779, Sigma-Aldrich, St. Louis, MO, USA). Afterwards livers were filtered through a 250 µm mesh. A gradient was applied by 35% Nycodenz (1002424, Axis-Shield, Alere Technologies, Oslo, Norway) and cells were separated. Utilizing anti-CD146 MicroBeads (ME-9F1, 130-092-007, Miltenyi Biotech, Bergisch Gladbach, Germany) LSECs were isolated by magnetic-activated cell sorting according to the manufacturers’ instructions. For additional analysis, LSECs were pooled from three mice.
Extracted molecule
total RNA
Extraction protocol
Using the Qiagen RNeasy Mini Kit RNA was extracted from retina samples according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol.
Hybridization protocol
Hybridization (16h x 45°C) was processed according to the standard Affymetrix protocol.
Scan protocol
Affymetrix GeneArray Scanner3000
Description
Gene expression data from isolated liver sinusoidal endothelial cells of mice with deficiency of Lyve-1, replicate 2.
Data processing
The data were analyzed with a commercial software called JMP Genomics, version 6, from SAS. Gene expression profiling was performed using arrays of mouse Mogene-2_0-type from Affymetrix. A Custom CDF Version 18 with Entrez based gene definitions was used to annotate the arrays. The Raw fluorescence intensity values were normalized applying quantile normalization, RMA background correction and Medianpolish Probeset Summary
