Supplementary Information for Attractive Interaction between Fully Charged Lipid Bilayers in a Strongly-Confined Geometry T. Mukhina 1,2 , A. Hemmerle 1,3 , V. Rondelli 4 , Y. Gerelli 2 , G. Fragneto 2 , J. Daillant 3 , T. Charitat 1 November 6, 2019 1 UPR 22/CNRS, Institut Charles Sadron, Universit´ e de Strasbourg, 23 rue du Loess, BP 84047 67034 Strasbourg Cedex 2, France 2 Institut Laue-Langevin, 71 av. des Martyrs, BP 156, 38042 Grenoble Cedex, France 3 Synchrotron SOLEIL, L’Orme des Merisiers, Saint-Aubin, BP 48, F-91192 Gif-sur-Yvette Cedex, France 4 Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Universit´ a degli Studi di Milano, LITA, Via F.lli Cervi 93, 20090 Segrate, Italy 1 Materials DPPS (L−α 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt)) and DSPC (L−α 1,2-distearoyl- sn-glycero-3-phosphocholine) were purchased from Avanti Polar Lipids (Lancaster, Alabama). Pure water (resistivity 18.2 MΩ.cm) was obtained from a Millipore purification system. D 2 O was supplied by the Institut Laue-Langevin (ILL, Grenoble, France). All the solvents used in the preparation were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and used without further purifications. 2 Sample preparation Supported bilayers were prepared on 25 cm 2 surfaces of n-doped silicon single crystals cut along the (111) direction. The surface roughness was lower than 5 Å, as determined by neutron reflectometry (NR) and X-ray reflectometry (XRR) experiments. Prior to their use, silicon blocks were cleaned using ultra-sonic bath in the following sequence of solvents: acetone, chloroform, ethanol and water. The blocks were sonicated in each solvent for 20 minutes. They were then dried under a nitrogen stream and treated with air-plasma (Harrick Plasma, US) for 2 minutes (for NR measurements) or with UV-Ozone (BioForce Nanosciences, USA) for 20 minutes (for XRR measurements) to make their surface as hydrophilic as pos- sible. Depositions of double bilayer systems were performed by Langmuir Blodgett (LB) and Langmuir Schaefer (LS) techniques using the 611 and 1212D NIMA Langmuir troughs (Coventry, UK) available at the Partnership for Soft Condensed Matter (PSCM, Grenoble, France). Monolayers at the air-water interface (prepared as described in [S1]) were compressed to a lateral pressure π = 40 mN/m. While the pressure was kept constant, the monolayer was transferred to the silicon block by LB. In the case of double bilayer systems this procedure was repeated three times while for triple bilayer systems it was repeated five times. In all cases, the final monolayer was deposited by a LS transfer. The samples were sealed inside a solid-liquid cell to be mounted on the neutron or x-ray reflectometers. The temperature of the cell was controlled using a water circulation bath. The water reservoir of the cell was connected to a HPLC pump in order to be able to change the subphase for the application of the contrast variation method [S2]. 1