GSM6482731: WT_24h rep1; Mus musculus; RNA-Seq
Total RNA was isolated from culture cells and purified using a Direct-zol RNA MicroPrep Kit (Zymo Research) as described by the manufacture.  mRNAs were enriched by incubation with Oligo d(T) Magnetic Beads (NEB). To fragment Poly A-enriched mRNA, mRNAs were incubated in 2X Superscript III first-strand buffer (Invitrogen) with 10 mM DTT at 94°C for 9 minutes.  The 10 µ l of fragmented mRNAs were incubated with 0.5 µl of Random primers (3 µg/µl) (Invitrogen), 0.5 µl of Oligo dT primer (50 µM) (Invitrogen), 0.5 µl of SUPERase-In (Ambion), and 1 µl of dNTPs (Invitrogen) at 50°C for 1 min.  After the incubation, 5.8 µl of water, 1 µl of DTT (100 mM), 0.1 µl of Actinomycin D (2 µg/µL), 0.2 µl of 1% Tween-20 and 0.5 µl of Superscript III (Invitrogen) were added and incubated on the following conditions: 25°C for 10 min, 50°C for 50 min, and 4°C hold.  The mixture was purified with RNAClean XP beads (Beckman Coulter) as described by the manufacture and eluted with 10 µl of water.  For second-strand synthesis with dUTP, the RNA/cDNA double-stranded hybrid was then added to 1.5 µl of Blue Buffer (Enzymatics), 1.1 µl of dUTP mix (10 mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 µl of RNAse H (5 U/µl) (Enzymatics), 1.05 µl of water, 1 µl of DNA polymerase I (Enzymatics), and 0.15 µl of 1% Tween-20.  The mixture was incubated at 16°C for 2.5 hr.  The resulting dUTP-marked dsDNA was purified with 2 µl of SpeedBeads (GE Healthcare) in 20% PEG 8000/2.5M NaCl (final 13% PEG 8000), and eluted with 40 µl of EB (Zymo Research).  The eluted dsDNA was carried out end repair by blunting, A-tailing and adapter ligation as previously described (Heinz et al., 2010) using barcoded adapters (Bioo Scientific).  The end repaired dsDNA was amplified for 16 cycles using 0.05 U/µl KAPA High Fidelity HotStart (Kapa Biosystems) and 1 µM each of primers Solexa 1GA and Solexa 1GB.  The amplified libraries were selected using PAGE/TBE gel (Invitrogen) for 225-325 bp fragments by gel extraction.