The amount of sequence obtained by modern sequencing machines greatly exceeds the sequencing depth requirements of many experiments, especially those involving organisms with small genomes. In the interest of economy and efficiency, various strategies have been developed for multiplexing, in which samples are uniquely tagged with short identifying sequences known as barcodes, pooled, and then sequenced together in a single lane. The resulting combined sequence data are subsequently sorted by barcode before bioinformatic analysis. This unit contains a barcoding protocol for the preparation of up to 96 ChIP samples for multiplex sequencing in a single flow cell lane on the Illumina platform. This strategy may be extended to even larger numbers of samples and may also be generalized to other sequencing applications or sequencing platforms. © 2013 by John Wiley & Sons, Inc.