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The mRNA with polyA structure in the total RNA was enriched by Oligo(dT) magnetic beads, and the RNA was broken down to fragments of about 300bp in length by ion interruption. The 300 bp length fragment was chosen because the splice length is fixed and a shorter interrupted fragment length would result in a higher proportion of splice sequences, thus reducing the proportion of valid data; a longer interrupted fragment length would be detrimental to the generation of clusters during up-sequencing. The first strand of cDNA was synthesised using RNA as the template and a 6-base random primer and reverse transcriptase, and the first strand cDNA was used as the template for the second strand cDNA synthesis.
After library construction, PCR amplification was used to enrich the library fragments, followed by library selection based on fragment size, which was 450 bp. The libraries were then quality checked by Agilent 2100 Bioanalyzer, and the total library concentration and the effective library concentration were tested. The libraries were then mixed in proportion to the effective library concentration and the amount of data required for the library, with the libraries containing different Index sequences (each sample with a different Index, and finally the downstream data for each sample based on the Index). The mixed libraries are uniformly diluted to 2nM and denatured by alkali to form single-stranded libraries.
After RNA extraction, purification and library construction, the libraries were sequenced using Next-Generation Sequencing (NGS) technology, based on the Illumina sequencing platform, for paired-end (PE) sequencing. |
RNA-Seq |
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PCR |
PAIRED
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