Truseq rna sample preparation v2 low sample protocol guide

For each genotype, 1 μg of RNA from each of the four biological replicates was pooled (4 μg) into a single sample and used for cDNA library preparation. The cDNA libraries were prepared following the TruSeq RNA Sample Preparation v2 low sample (LS) protocol guide (Illumina Inc.). Poly(A)-containing mRNA was purified twice using poly(T) oligonucleotide-attached magnetic beads. In the second elution, the Poly(A) RNA was fragmented and primed for cDNA synthesis at 94°C in an attempt to obtain a median insert size of 180 bp fragment. The fragmented RNA templates were primed with random hexamers, and the first strand was synthesized by four cycles of 25°C for 10 minutes, 42°C for 50 minutes, and 70°C for 15 minutes. Following second strand synthesis (16°C for one hour), end repair was performed to generate blunt ends followed by adenylating of the 3′ blunt-ended double-stranded cDNAs to allow for subsequent ligation of multiple indexing adaptors. cDNA fragments were amplified and enriched using 15 cycles of PCR according to Illumina TruSeq RNA Sample Prep v2 LS protocol. The libraries were quantified using a Qubit^® 2.0 Fluorometer (Life Technologies), and the quality was analyzed with the TapeStation 2200 (Agilent) using the D1K tape for validating the purity and estimating the insert size.