GSM8442933: RKO-dox-Cas9, sgMAGOH-1, nucleus, biol rep 3; Homo sapiens; RNA-Seq
For subcellular fractionation, ~2x10^6 cells were gently lysed in REAP buffer (0.1% NP-40 in 1x PBS) at 4°C. Nuclei and cytoplasmic fractions were separated through centrifugation at 3k g for 60 sec at 4°C twice. RNA isolation was done with High Performance RNA Isolation kit (Vienna, VBC Molecular Tools Shop, RNAisomag) and a KingFisher Flex robot (Thermo Fisher Scientific). Generation of 3' mRNA sequencing libraries was done according to manufacturer's recommendations (QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina, UMI Second Strand Synthesis Module for QuantSeq FWD, and PCR Add-on Kit for Illumina, Lexogen).