GSM1808857: RNASeq DC +IL10 4h; Mus musculus; RNA-Seq
Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen). Double-stranded cDNA was synthesized with random hexamer primers, SuperScript II, DNA polymerase I, and T4 DNA polymerase (Invitrogen) and fragmented by Bioraptor. After end repair (Epicentre DNA END-Repair kit) and the addition of an “A” nucleotide to the 3’ ends with Taq DNA polymerase, two mixed Solexa adaptors (Illumina) were ligated to DNA ends using T4 DNA ligase (New England Biolabs). 250-500 bp fragments were isolated on 2% E-Gels (Invitrogen) and amplified for 18 cycles using PE 1.0 and 2.0 primers (Illumina) and Phusion High Fidelity PCR Master Mix (New England Biolabs). PCR products were run on a gel, and 250-500 bp fragments were purified. Final PCR products were sequenced on an Illumina HiSeq 2000 platform.