Barcode lentiviral vector, sequencing and analysis workflow. (a) The 20 bp DNA barcode was cloned into the non-coding region of a SIN (self-inactivating) lentiviral vector upstream of a UBC-eGFP cassette. The P5 Illumina sequencing adapter sequence was integrated next to the barcode, and the P7 adapter was added during the PCR amplification step (primer positions shown). (b) This PCR results in a 250 bp fragment that includes a 4 bp indexing tag to allow pooling of multiple samples into a single lane of a flow cell, in addition to the 20 bp random barcode sequence, and flanked on either side by eight 'anchor' bases, which act as markers to identify true barcode sequences within the sequencing data. Finally, the fragments contain a spacer of approximately 90 bp and the second (P7) Illumina adapter for sequencing. Integrating the adapter into the barcode vector allows for single-end 36 bp (short) sequencing reads in which the barcode end is always sequenced. (c) Data analysis workflow.