Directional RNA sequencing
Directional RNA-seq samples were prepared according to a slightly modification of the protocol provided by Illumina. Briefly, cDNA libraries were prepared starting from 5 ?g of total RNA as follows. First, total RNA was selected twice with Sera-Mag Magnetic Oligo(dT) Beads (Thermo Scientific) to isolate poly(A)+ RNA.  The amount of rRNA was monitored to be less than 2% in each poly(A)+ RNAs by using a Total RNA Pico Bioanalyzer chip (Agilent). poly(A)+ RNA was fragmented by heating at 94?C for 3 min in 1 x fragmentation buffer (Affymetrix), followed by ethanol precipitation. Fragmented RNA was decapped with TAP, followed by extraction with PCI and ethanol precipitation. Fragmented and decapped RNA was 3?-dephosphorylated using Antarctic phosphatase (New England Biolabs (NEB)). The RNA was 5?-phosphorylated using T4 polynucleotide kinase (NEB). The modified RNA was cleaned up with an RNeasy MinElute kit (Qiagen). The RNA was ligated to 1 x v1.5 sRNA 3? adaptor (Illumina) with T4 RNA ligase 2, truncated K277Q (NEB) at 4?C overnight. This RNA was ligated to SRA 5? adaptor (Illumina) with T4 RNA ligase (Illumina) at 20?C for 1 hr. cDNA was synthesized with specific RT primer and SuperScriptIII First-Strand Synthesis System (Life Technologies Co.). Before the amplification, two cDNA replicates were prepared. Each cDNA was amplified with Phusion DNA Polymerase (FINNZYMES). Thermal-cycling conditions were as follows: 30 sec at 98?C, 12 cycles of 98?C for 10 sec, 60?C for 30 sec, and 72?C for 15 sec, followed by 10 min at 72?C. The PCR product was purified twice with AMPure XP (Beckman Coulter) to generate a library and analyzed on a DNA1000 Bioanalyzer chip (Agilent) for precise quantification of molarity. After confirmation of the high quality of the cDNA library samples, we sequenced them for 51 bp single-end read using the Illumina HiSeq 2000 per each sample with the small RNA sequencing primer (Illumina) according to the manufacturer?s instructions.