title: Illumina NovaSeq 6000 paired end sequencing; RNA-seq of MUTZ3 cells in experiments to investigate EVI1 regulation
design: RNA-seq of MUTZ3 cells in experiments to investigate EVI1 regulation
library_construction_protocol: MUTZ3 cells were harvested at the indicated time point after treatment. The MUTZ3 cell lines were cultured in αMEM (HyClone) with 20% fetal calf serum (FCS) and 20% conditioned 5637 medium. They were supplemented with 50 U/mL penicillin and 50 μg/mL streptomycin.  The sgRNAs were either cloned into pLentiV2_U6-IT-mPgk-iRFP720 using BsmBI restriction sites, px330 using BbsI or were in vitro transcribed using the T7 promoter. Lentiviruses were prepared by transfecting 293T cells with lentiviral packaging constructs pSPAX2 / pMdelta2.G and sgRNA cloned into pLentiV2_U6-IT-mPgk-iRFP720.  RNA was isolated either using Trizol or the Qiagen Allprep DNA/RNA kit and protocol (Qiagen, #80204).  For RNA sequencing, sample libraries were prepped using 500 ng of input RNA according to the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche) using Unique Dual Index adapters (Integrated DNA Technologies, Inc.).