GSM8864785: DMSO control well, biol repl 17; Homo sapiens; RNA-Seq
Cell culture media was aspirated using a STARlet liquid handler (Hamilton) and lysis buffer was dispensed directly into each well with a Multidrop Combi Reagent Dispenser (ThermoFisher Scientific) and placed on a shaker for 15 minutes at 900 rpm. Libraries were prepared using a customized version of the DRUG-seq protocol. Cell lysate was transferred into 384-well PCR plates and reverse transcription (RT) mix was dispensed into each well. Using an Echo 650 Liquid Handler (Beckman Coulter), barcoded RT primers were dispensed into each well. Plates were incubated at 42°C for 2 hours. Following RT, each well was pooled into a single tube and purified using DNA Clean & Concentrator-100 (Zymo Research) followed by concentration with Agencourt RNAClean XP beads (Beckman Coulter). Single-stranded DNA was removed by incubating cDNA with Exonuclease I (New England Biolabs) followed by pre-amplification of the cDNA with DRUG-seq PCR primers. cDNA was fragmented and tagged with Illumina sequencing adapters using Tagmentation DNA Enzyme (TDE1). A final PCR was performed to amplify the libraries and add P7, P5, and index sequences.