mRNA Extraction and RNA-seq Library Preparation

The mRNA extraction, cDNA library construction and sequencing were performed by the Beijing Center for Physical and Chemical Analysis (BCPCA) (Beijing, China). Total RNA was extracted from each sample using the TRlzol reagent and digested with DNase I. Oligo (dT) magnetic beads were used to enrich mRNA from the total RNA and then broken into short fragments by a fragmentation buffer. To build the cDNA libraries based on the four samples (Solexa/Illumina 2,500 platform: 101 bp short insert-paired end), we followed the methods of Neeraja et al. (2012 (link)) and Tang et al. (2011 (link)).

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Zhang J., Li X., Lu F., Wang S., An Y., Su X., Li X., Ma L, & Han G. (2017). De novo Sequencing and Transcriptome Analysis Reveal Key Genes Regulating Steroid Metabolism in Leaves, Roots, Adventitious Roots and Calli of Periploca sepium Bunge. Frontiers in Plant Science, 8, 594.

Publication 2017

2,500 platform

Buffer Cdna libraries Dnase i Mrna Oligo Physical

Corresponding Organization : Tianjin University of Science and Technology

Other organizations : Tianjin University of Traditional Chinese Medicine, Beijing Academy of Science and Technology

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Variable analysis

independent variables

MRNA extraction method (TRIzol reagent)
CDNA library construction method (Neeraja et al. 2012 and Tang et al. 2011)

dependent variables

Sequencing of cDNA libraries (Solexa/Illumina 2,500 platform: 101 bp short insert-paired end)

control variables

Total RNA digestion with DNase I
Enrichment of mRNA from total RNA using oligo (dT) magnetic beads
Fragmentation of mRNA into short fragments

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