Nextera enzyme

Cells attached to wells were lysed directly in wells with 15 μl lysis buffer. Then plates were sealed and placed on a microplate shaker for 15 min at 900 rpm. Barcoded DRUG-seq RT primers (Supplementary Data 1) were dispensed into individual wells with an Echo liquid handler (Labcyte Inc), and cell lysate was transferred into 384-well PCR plates pre-dispensed in each well with RT mix and diluted ERCC mix1 (Thermo Fisher). Plates were incubated at 42 °C for 2 h. Material from each well in a 384-well plate was pooled into a single sample, purified with DNA clean & concentrator-100 kit (Zymo Research) and Agencourt RNAClean XP beads (Beckman Coulter). After ExoI treatment, material was pre-amped with DRUG-seq PCR primers and purified. The pre-amped material was fragmented with Nextera enzyme (Illumina), and individual libraries were indexed and quantitated with qPCR before sequencing on a Hiseq 4000 (Illumina). See Supplementary Information for specific barcode and primer sequences and detailed protocol.

Bench-top library construction was done following the Illumina Nextera protocol for tagmentation. 5 μl of 4.8 ng μl⁻¹ (24 ng total) of purified gDNA was mixed with 15 μl of Nextera enzyme (Illumina), 5 μl of 5 × Tagmentation buffer (50 mM Tris-HCl pH 8.0 (Sigma), 20 mM MgCl₂ (Sigma)) and 10 μl of H₂O, then incubated for 10 min at 58 °C. 2.5 μl of stop solution (2.5% wt per vol SDS (Sigma) in H₂O) was added to the mixture, followed by incubation for 10 min at 72 °C. Tagmented DNA was purified by mixing with 49.5 μl SPRI bead suspension (Beckman-Coulter), binding for 10 min at 25 °C, magnetically separating the beads, washing twice with ethanol and eluting the product off the beads with 50 μl of 10 mM Tris-HCl pH 8.0.