BmN4-1_MatePair
Two hundred micrograms of the total RNA obtained was subjected to oligo-capping, with some modifications from the original protocol.  Briefly, after successive treatment of the RNA with 2.5 U of BAP (TaKaRa) at 37?C for 1 h and 40 U of TAP (Ambion) at 37?C for 1 h,  the BAP?TAP-treated RNAs were ligated to 1.2 ?g of the RNA oligonucleotide 5?-AGCAUCGAGUCGGCCUUGUUGGCCUACUGGCAGCAG -3?  using 250 U ofT4 RNA ligase (TaKaRa) at 20?C for 3 h. After DNase I treatment (TaKaRa), poly(A)-containing RNA was selected using oligo-dT powder (Collaborative). The first strand cDNA was synthesized with 10 pmol of dT primer (5?-GCGGCTGAAGACGGCCTATGTGGCC(T)17-3?) using SuperScript II (Invitrogen), with incubation at 42?C overnight. The template RNA was degraded by alkaline treatment.  For PCR, 20% of the first strand cDNA was used as the PCR template. Gene Amp PCR kits (PerkinElmer) were used with the PCR primers 5?-AgCATCgAgTCggCCTTgTTg-3? and 5?-GCGGCTGAAGACGGCCTATGT -3? under the following reaction conditions:  15 cycles at 94?C for 1 min, 56?C for 1 min, and 72?C for 10 min. This primer, 5?-AgCATCgAgTCggCCTTgTTg-3?, is biotinylated with three 10th,16th,17th T. The PCR fragments were size-fractionated by 1% agarose gel electrophoresis, and the fraction that contained the 0.5-5 kbp fragments was recovered.  The quality and quantity of the obtained cDNAs were assessed using a BioAnalyzer (Agilent) 600ng of the size-fractionated cDNA was end repaired with T4 DNA Polymerase, T4 Polynucleotide Kinase and Klenow DNA Polymerase. These enzymes are supplied with the Mate Pair Library Sample Preparation Kit (illumina).  End repaired DNA are fragmented with nebulization 32psi for 6minutes. Nebulizers and nebulization buffer are supplied by illumine.  Fragmented DNA are treated with EcoP15I (New England BioLabs) at 37?C for 1h.  After Purifying biotinylated DNA the End Repair, A-tail DNA Fragmens, Ligate Adapters should be done as illumine protocol. Then using PCR we can enrich Adapter-Modified Fragments. The PCR fragments were size-fractionated by 8% polyacrylamide gel electrophoresis, and the fraction that contained the 260?280-bp fragments was recovered. The quality and quantity of the obtained DNAs were assessed using a BioAnalyzer (Agilent).  One nanogram of the size-fractionated DNA was used for sequencing reactions with the Illumina Hiseq 2000.  The sequencing reactions were performed according to the manufacturer's instructions. 