title: Single-tube linear DNA amplification for genome-wide studies using a few thousand cells.
abstract: Linear amplification of DNA (LinDA) by T7 polymerase is a versatile and robust method for generating sufficient amounts of DNA for genome-wide studies with minute amounts of cells. LinDA can be coupled to a great number of global profiling technologies. Indeed, chromatin immunoprecipitation coupled to massive parallel sequencing (ChIP-seq) has been achieved for transcription factors and epigenetic modification of chromatin histones with 1,000 to 5,000 cells. LinDA largely simplifies reChIP-seq experiments to monitor co-binding at chromatin target sites. The single-tube design of LinDA is ideal for handling ultrasmall amounts of DNA (<30 pg) and is compatible with automation. The actual hands-on working time is less than 6 h with one overnight reaction. The present protocol describes all materials and critical steps, and provides examples and controls for LinDA. Applications of LinDA for genome-wide analyses of biobank samples and for the study of chromatin conformation and nuclear architecture are in progress.
authors.affiliation: Department of Cancer Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg-Illkirch, France. shankar@igbmc.fr
authors.forename: Pattabhiraman
authors.initials: P
authors.lastname: Shankaranarayanan
authors.forename: Marco-Antonio
authors.initials: MA
authors.lastname: Mendoza-Parra
authors.forename: Wouter
authors.initials: W
authors.lastname: van Gool
authors.forename: Luisa M
authors.initials: LM
authors.lastname: Trindade
authors.forename: Hinrich
authors.initials: H
authors.lastname: Gronemeyer
mesh_terms: D001482:Base Composition; D047369:Chromatin Immunoprecipitation; D023281:Genomics / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques; D010963:Plasmodium falciparum / Q000235:genetics; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic