BmN4_TSS
BioProject of this experiment was changed from PRJDB1760 to PRJDB1759.
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 degree_C for 1 h and 40 U of TAP (Ambion) at 37 degree_C for 1 h, the BAPmicroTAP-treated RNAs were ligated to 1.2 microg of the RNA oligonucleotide 5micro-ACCGAGAUCUACACUCUUUCCCUACACGACGCUCUUCCGAUCUGG-3micro using 250 U ofT4 RNA ligase (TaKaRa) at 20 degree_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 random hexamer primer (5micro-GGUCUAGCCUUCUCGCAGCACAUCCCUUU-3micro) using SuperScript II (Invitrogen), with incubation at 12 degree_C for 1 h and 42 degree_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 5micro-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3micro and 5micro-TCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGCTAGAGCATACGGCAGAAGACGAAC -3micro under the following reaction conditions: 15 cycles at 94 degree_C for 1 min, 56 degree_C for 1 min, and 72 degree_C for 2 min. The PCR fragments were size-fractionated by 12% polyacrylamide gel electrophoresis, and the fraction that contained the 150micro250-bp fragments was recovered. The quality and quantity of the obtained single-stranded first-strand cDNAs were assessed using a BioAnalyzer (Agilent). One nanogram of the size-fractionated cDNA was used for sequencing reactions with the Illumina GA. The sequencing reactions were performed according to the manufacturer's instructions.