GSM7698077: RNA expression in T-ALL PDX line with 300nM Nav and 1.5uM Ida, biological replicate 2; Homo sapiens; RNA-Seq
After culturing for 16 hours cells in drug, RNA was extracted using the Monarch Total RNA Miniprep Kit (NEB). Three replicate samples were treated and isolated. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 500ng to 1ug of total RNA. Ribosomal RNA was blocked using FastSelect reagents (Qiagen 334385) during cDNA synthesis. RNA was fragmented in reverse transcriptase buffer with FastSelect reagent and heating to 94 degrees for 5 minutes, 75 degrees for 2 minutes, 70 degrees for 2 minutes, 65 degrees for 2 minutes, 60 degrees for 2 minutes, 55 degrees for 2 minutes, 37 degrees for 5 minutes, 25 degrees for 5 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.