STID3042_H51
Total RNA was prepared in accordance with Illumina's sample preparation protocol for PE sequencing of mRNA. In brief, 5–10 micrograms of total RNA was fragmented by heat between 2 and 3 min based on the desired insert size, reverse-transcribed using Superscript II (Invitrogen), and transformed to double-stranded cDNA. To improve PE RNA-seq data quality, we introduced an additional gel-based size selection step after cDNA double-strand synthesis and before the ligation of the PE adapters. We also integrated the use of T4 ligase (Enzymatics Inc.) to improve the efficiency of adapter ligation. The gel dissolutions of all gel-based purification steps were conducted at room temperature under slight agitation. After the enrichment of cDNA template by PCR, the concentrations and the sizes of the libraries were measured using a Qubit fluorometer (Invitrogen) and DNA 1000 kit (Agilent Technologies) on an Agilent 2100 Bioanalyzer, respectively.
WCMC-MR
STID3042_H51