title: GSM8625262: MDA-MB-231 CDK4/6i (biol rep 2); Homo sapiens; RNA-Seq
library_construction_protocol: MDA-MB-231 cells expressing with mCerulean-tagged Geminin (a.a 1−110) were treated with the indicated drugs for 42 hr. Cells were then dissociated, and mCerulean-positive cells were isolated by FACS. Collected cells were immediately lysed in TRIzol LS Reagent (Invitrogen, 25 #10296010) with an additional 0.2 ml of chloroform per 1ml of TRIzol to facilitate phase separation. Following centrifugation at 12,000 rpm for 15 min at 4°C, the aqueous phase was transferred and RNA was precipitated by adding 0.5 ml of isopropyl alcohol (per 1 ml of TRIzol) along with Glycoblue (50 µg/ml) (Applied Biosystems, AM9515), then incubated overnight at - 35 20°C. Precipitated RNA was centrifuged at 12,000 rpm for 10 min at 4°C, the supernatant was discarded, and the RNA pellet was washed with 75% ethanol. After a final centrifugation at 7,500 rpm for 5 min at 4°C, the RNA pellet was air-dried and resuspended in RNase-free water. cDNA synthesis was carried out using the Clontech Ultra Low v4 kit, followed by library construction with NexteraXT.