GSM8386594: Euseq_mESC_G2_M_rep2; Mus musculus; OTHER
RNA samples were then precipitated by adding 350 µL of water, 16 µL of 5M NaCl, 1 µL of glycogen and 832 µL of cold ethanol, incubating the mix at -70ºC and centrifuging for 30-60 minutes at 4ºC, 16000 G. The RNA pellet was washed twice in a cold solution of 75% ethanol/water, centrifuged and the pellet resuspended in 400 µL of water. Biotinylated RNA was captured using 200 µL of Dynabeads MyOne streptavidin C1 (Invitrogen, 65001) per sample. The beads had been previously washed thrice in buffer W1x (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.5% Tween-20), twice in Solution A (0.1M NaOH, 0.05M NaCl in water) and twice in Solution B (0.1M NaCl in water). The volume of solutions used for washing the beads was calculated as 200 µL per sample and scaled up depending on the total number of samples. After the last wash, the beads were removed from the magnet and resuspended in buffer W2x (same as W1x but two times concentrated) in two times the original volume (400 µL per sample). 400 µL of this washed bead mix were added to each biotinylated RNA sample and incubated 30 minutes at room temperature on a rotating wheel. Afterwards, the bead-RNA samples were washed three times with 800 µL of buffer W1x. Each Biotinylated-RNA sample was eluted by resuspending the beads in a solution containing 55 µL of 10mM Tris pH 8 and 1.1 µL of 2-mercaptoethanol and incubating them for one hour at room temperature. RNA libraries were generated without the ribodepletion step and sequenced at the Genomics platform of the University of Geneva.