To measure the 1,000 landmark transcripts, we adapted a method involving ligation-mediated amplification (LMA) followed by capture of the amplification products on fluorescently-addressed microspheres (Peck et al., 2006). We extended this method to a 1,000-plex reaction (Figure 1A; protocols at clue.io/sop-L1000.pdf). Briefly, cells growing in 384-well plates were lysed and mRNA transcripts captured on oligo-dT-coated plates. cDNAs were synthesized and subjected to LMA using locus-specific oligonucleotides harboring a unique 24-mer barcode sequence and a 5′ biotin label. The biotinylated LMA products were detected by hybridization to polystyrene microspheres (beads) of distinct fluorescent color, each coupled to an oligonucleotide complementary to a barcode, and then stained with streptavidin-phycoerythrin. Thus, each bead was analyzed both for its color (denoting landmark identity) and fluorescence intensity of the phycoerythrin signal (denoting landmark abundance). Because only 500 bead colors are commercially available, we devised a strategy that allows two transcripts to be identified by a single bead color (STAR Methods and Figure 1B). 955 shRNAs targeting landmark transcripts were used to empirically validate L1000 probes (Figure 1C). The final assay, which we call L1000, contains
