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Total RNA was extracted using Trizol reagent kit according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads, while prokaryotic mRNA was enriched by removing rRNA by Ribo-ZeroTM Magnetic Kit. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit, end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel lectrophoresis, PCR amplified, and sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology Co. |
RNA-Seq |
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PolyA |
PAIRED
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