RNA extraction was carried out using the NucleoSpin RNA II kit (740955.50; MACHEREY-NAGEL, Düren, Germany). cDNA synthesis from 1 μg total RNA as control for RNA-seq was carried out using the iScript cDNA synthesis kit (Bio-Rad; 170-8891, CA, Hercules, USA). Starting amount of RNA-seq sample was 1 μg. rRNA depletion was performed with the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat; cat. no. MRZH11124, Epicentre, Madison, WI, USA) according to the manufacturer's instructions. RNA fragmentation reactions were performed using fragmentation buffer (5×; 200 mM Tris-Ac, 500 mM Potassium-Ac, 150 mM Magnesium-Ac) in a final concentration of 1× per reaction. Each 50 μl fragmentation reaction was incubated at 95°C for 1.5 minutes on a thermal cycler, and placed on ice for 10 minutes. Ethanol precipitation was used to purify the reactions. The fragmented rRNA depleted RNA was combined with 5 μg of random hexamers (Roche) in a final volume of 11 μl, incubated at 65°C for 5 minutes, and afterwards immediately placed on ice. The remaining reagents were added to the reaction: 4 μl 5× First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl dNTP mix (10 mM Invitrogen), 2 μl Actinomycin D (0.1 μg/ μl Sigma), 1 μl of RNase H (40U/ml Ambion), and 1 μl SuperScript III (200 U Invitrogen). The first strand reaction was incubated at 25°C for 10 minutes, 50°C for 90 minutes, followed by deactivation at 70°C for 15 minutes, followed by MinElute Reaction Cleanup Kit (Qiagen), according to the manufacturer's protocol. The second strand was synthesized by adding 20 μl 5x Second strand buffer (Invitrogen), 4 μl 5x First strand buffer (Invitrogen), 2 μl DTT (0.1 M Invitrogen), 1 μl random hexamers (5 mg/ml Roche), dUTP mix (12.5 mM Invitrogen), 1 μl RNase H (8U/ml Ambion), 1 μl E. coli DNA polymerase I (10U/ μl Invitrogen), and 1 μl E. coli DNA ligase (10 U/μl NEB) to the purified sample (100 μl total volume). After 2 hours at 16°C, 1 μl of T4 DNA polymerase (10U/μl Promega) was added and the reaction was incubated at 16°C for 10 minutes. The ds-cDNA was purified using the MinElute Reaction Cleanup Kit (Qiagen), according to the manufacturer's protocol. DNA samples were prepared for sequencing by end repair of 5 ng total cDNA as measured by Qubit (Invitrogen). Adaptors were ligated to the DNA fragments, followed by a pre-PCR of 4 cycles, size selection (∼300 bp) and subsequently 11 cycles of PCR amplification.
GEO Accession
GSM1446882