title: GSM5979967: R254AKlf3MEFs RNA-seq Rep 2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from MEF wildtype KLF3 cells and MEFs R254A mutant KLF3 cells with Tri-reagent according to the manufacturer's protocol (Qiagen, Cat#74004). 200 mL of chloroform was added to 1 mL Tri-reagent and vortexed for 10 seconds. The mixture was spun at 12,000 x g for 5 minutes. The aqueous phase was transferred into a new Eppendorf tube. The aqueous phase was mixed with  an equal volume of ethanol. The RNA-ethanol mixture was added to the column and washed once with wash buffer. RNA retained in the column was treated with DNA removal kit (Qiagen, Cat#79254) to ensure the purity of RNA samples. After DNase treatment, bound RNA was washed with wash buffer and then eluted with RNase A free water.  RNA concentration was determined by UV-light absorbance at 260 nm using a Nanodrop machine (Thermo Fisher Scientific). The integrity of RNA was checked by Bioanalyzer (Agilent). TruSeq Stranded mRNA