title: Illumina HiSeq 4000 sequencing; RNA-seq of L3.6pl cells treated with CI994, TW9, and CI994 + JQ1
design: RNA-seq of L3.6pl cells treated with CI994, TW9, and CI994 + JQ1
library_name: L3.6pI_CI994_2_s
library_construction_protocol: Both JQ1 and TW9 were synthesized in and acquired from the Stefan Knapp lab (Goethe University, Frankfurt am Main). CI994 is from Selleck Chemicals, and its cat.no. is S2818. Cells were treated at 100nM for all compounds for 24 hours before harvesting. Cells were treated for 24 hours before harvesting. RNA was isolated with QIAzol Lysis Reagent (Qiagen) added to the cells and centrifuged at full speed for 30 min. The aqueous phase was removed and mixed with equal volume of chloroform and centrifuged again for 20 minutes. The aqueous phase was then removed and the RNA was allowed to precipitate in isopropanol and incubated at -20°C overnight. Subsequently, RNA was washed twice with 70% ethanol and re-suspended in RNase free water.  The integrity of RNA was validated by gel electrophoresis and 500ng were used to make the libraries in triplicates for each condition. Libraries were made using the TruSeq RNA Library Prep Kit V2 (Illumina) according to the manufacturer's instructions.