title: GSM8093931: membrane, 240min, replicate1; Mus musculus; RNA-Seq
library_name: GSM8093931
library_construction_protocol: Cells were harvested at 0, 30, 60, 120 and 180 min after addition of flavopiridol followed by cell fractionation. Briefly, medium was aspirated, 5 ml of ice-cold PBS supplemented with 100 µM cycloheximide (A0879,0001, Biochemika) was added to the plates, cells were scraped from the plates, transferred to 15ml falcon and spun down. Pellet was resuspended in 500 µl of ice-cold permeabilization buffer (110 mM KOAc, 25 mM K-HEPES pH 7.2, 2.5 mM Mg(OAc)2, 1 mM EGTA with freshly added 0.015% digitonin, 1 mM DTT, 100 μg/ml cycloheximide, 1X Complete Protease Inhibitor Cocktail and 40 U/mL RNaseOUT™). 100 µl of the sample was taken aside as Total extract and rest was incubated for 10 min at 4°C with rotation, followed by centrifugation at 3000 g 5 min at 4°C. Supernatant (corresponding to the Cytosolic fraction) was transferred to new tube while the pellet was resuspended in 5ml of wash buffer (110 mM KOAc, 25 mM K-HEPES pH 7.2, 2.5 mM Mg(OAc)2, 1 mM EGTA with freshly added 0.004% digitonin, 1mM DTT, 100μg/ml cycloheximide) and spun down again at 3000 g 5 min at 4°C. After centrifugation washed pellet was mixed with 500 µl of ice-cold lysis buffer (400 mM KOAc, 25 mM K-HEPES pH 7.2, 15 mM Mg(OAc)2, 0.5% (v/v) NP-40 and freshly added 1 mM DTT, 100 μg/ml cycloheximide, 1X Complete Protease Inhibitor Cocktail, 40 U/mL RNase Out) and incubated for 5 min on ice followed by centrifugation at 3000 g 5 min at 4°C to collect the supernatant (corresponding to the Membrane fraction) and the pellet (insoluble and the nuclear fraction). For additional purity nuclei were loaded on 10% sucrose cushion in lysis buffer and centrifuged at 200 g 5 min at 4°C. The cytosolic and membrane fractions were clarified at 7500 g 10 min at 4°C to remove cell debris.Obtained fractions were mixed with Trizol LS and RNA was isolated following the manufacturer instructions. One microgram of total RNA was used as an input for TruSeq Stranded mRNA Library Prep Kit according to the instructions of the manufacturer. TruSeq Stranded mRNA Library Paired-End