RNA-seq and read mapping

RNA extraction and quantitative real-time PCR

Total RNA was extracted using TRIZOL Reagent (Life technologies, Carlsbad, CA), and reverse transcription was performed using an Advantage RT-for-PCR Kit (Clontech Laboratories, Mountain View, CA) according the manufacturer’s instructions. For qPCR analysis, aliquots of double-stranded cDNA were amplified using a SYBR Green PCR Kit (Life technologies, Carlsbad, CA) and an ABI PRISM 7900 Sequence Detector. 

RNA sequencing and read mapping.

Total RNA was isolated using the mirVana micro­RNA isolation kit (Ambion, Austin, TX, USA) and treated with the DNA-free kit (Ambion) for the removal of contaminating genomic DNA. Poly(A)+ RNA was purified using a Dynabeads mRNA purification kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Approximately 100 ng of mRNA was fragmented by incubation for 5 min at 94 °C in 5× Array Fragmentation Buffer (Ambion). Double-stranded cDNA were synthesized using the SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen) using random hexamers. The reaction was purified using a QiaQuick PCR column (Qiagen, Valencia, CA). The Klenow 3′→5′ exo polymerase (NEB, Ipswich, MA) was used to add a single adenine base to the 3′ end of the blunt phosphorylated DNA frag­ments. After purification, Illumina PE Adapters (Illumina, San Diego, CA) were ligated to the ends of the DNA fragments using the Quick Ligation Kit (NEB, Ipswich, MA). DNA fragments were excised ranging from approximately 280 bp to 300 bp in length from a 2% low-melting agarose gel. The fragments were enriched by ten thermocycles using AccuPrime Pfx DNA Polymerase (Invitrogen). The PCR product was run on a Novex 8% Tris-borate-EDTA polyacrylamide gel (Invitrogen) and stained with SYBR Gold (Invitrogen). Gel slices containing the 340- to 360-bp fragments were excised and purified using the QiaQuick Gel Extraction Kit (Qiagen). Cluster generation and sequencing were conducted using the Standard Cluster Generation kit v4 and the 36-Cycle Sequencing kit v3 on the Illumina Cluster Station and GAIIx following the manufacturer’s instructions. Raw data from the GAIIx were analyzed using the Illumina Real Time Analysis (RTA) v1.6 software. A ΦX174 control lane was included in each Solexa run for matrix, phasing and error rate estimations as recommended by the manufacturer. The error rate of the ΦX174 control error rate was <0.28% for all of the sequencing runs. After removing the ribosomal RNA sequences from the GA reads, high-quality reads were aligned against the human genome assembly (NCBI build 37.1/hg19) using TopHat v1.0.14 with the RefSeq refGene annotation, which was downloaded from the UCSC Genome Browser. The mapping results were then processed with custom scripts and visualized on the UCSC Genome Browser as a custom track.