GSM5379810: RNA_ERHoxB8_WT_rep1; Mus musculus; RNA-Seq
Total RNA was isolated from in vitro culture cells and purified using a Direct-zol RNA MicroPrep Kit (Zymo Research) according to the manufacturer's instructions (Zymo Research). Poly A enriched mRNA was fragmented, in 2x Superscript III first-strand buffer with 10mM DTT (Thermo Fisher Scientific), by incubation at 94°C for 9 minutes, then immediately chilled on ice before the next step. The 10 µL of fragmented mRNA, 0.5 µL of Random primers (3 µg/µL) (Thermo Fisher Scientific), 0.5 µL of Oligo dT primer (50 µM) (Thermo Fisher Scientific), 0.5 µL of SUPERase-In (Ambion), 1 µL of dNTPs (10 mM) and 1 µL of DTT (10 mM) were heated at 50°C for one minute. At the end of incubation, 5.8 µL of water, 1 µL of DTT (100 mM), 0.1 µL Actinomycin D (2 µg/µL), 0.2 µL of 1% Tween-20 (Sigma) and 0.5 µL of Superscript III (Thermo Fisher Scientific) were added and incubated in a PCR machine using the following conditions: 25°C for 10 minutes, 50°C for 50 minutes, and a 4°C hold. The product was then purified with RNAClean XP beads (Beckman Coulter) according to manufacturer's instruction and eluted with 10 µL nuclease-free water. 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), 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 hours. The resulting dUTP-marked dsDNA was purified using 28 µL of SpeedBeads (GE Healthcare), diluted with 20% PEG8000, 2.5M NaCl to final of 13% PEG, eluted with 40 µL EB buffer (10 mM Tris-Cl, pH 8.5) and frozen at -80°C. The purified dsDNA (40 µL) underwent end repair by blunting, A-tailing and adapter ligation using barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR-amplified for 16 cycles, size selected by gel extraction.