Dna clean concentrator 100 kit

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.

Oligopaint libraries were purchased from CustomArray (Bothell, WA) and amplified using in vitro transcription as previously described [24 ]. Briefly, for each sub-pool to be amplified, we first optimized the template and primer concentrations using real-time PCR. We next performed large-scale limited-cycle PCR, and then used the product as template for in vitro transcription by T7 RNA polymerase (NEB, E2040S). The resulting ssRNA was then reverse transcribed (EP0752, ThermoFisher Scientific). RNA was digested by adding a mixture of 0.5 M NaOH and 0.25 M EDTA of equal volume to the reverse transcription reaction at 95° C for 10 minutes. Oligopaint ssDNA oligo probes were purified using the DNA Clean & Concentrator–100 kit (Zymo Research, Irvine, CA) paired with an EZ-Vac Vacuum Manifold (Zymo Research).