GSM2486568: G3; Apis mellifera; RNA-Seq
Samples for the analysis were transferred into liquid nitrogen and used for total RNA extraction using Tri-reagent (Ambion® Inc.). RNA was processed using the TruSeq mRNAseq sample prep kit (Illumina, Inc., CA, USA), starting from 2 micrograms of total RNA. The standard mRNA sample prep from Illumina was used to produce 36 base pairs long tags, 25-30 million per sample. Libraries were prepared according to Illumina's instructions accompanying the TruSeq RNA Sample Prep Kit v2 (Part #15026495). Briefly, 0.1–1 μg of total RNA were purified using oligo-dT attached magnetic beads using two rounds of purification. During the second elution of the polyA RNA, the  RNA is also fragmented. Cleaved fragments are then primed with random hexamers into first strand cDNA using reverse transcriptase and random primers. Overhangs resulting from fragmentation are converted into blunt ends using an End Repair Mix. The 3' to 5' exonuclease activity of this mix removes the 3' overhangs and the polymerase activity fills in the 5' overhangs. A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single ‘T’ nucleotide on the 3’ end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensures a low rate of chimera (concatenated template). formation. PCR is used to enrich selectively DNA fragments that have adapter molecules on both ends, and the DNA  amount in the library is amplified. The PCR is performed with a PCR Primer Cocktail that anneals to the ends of the adapters. Library is validated by using Agilent Technologies 2100 Bioanalyzer using the Agilent DNA 1000 DNA-specific chip. Cluster density is optimized by quantify libraries using PCR according to the Illumina Sequencing Library qPCR Quantification Guide (part # 11322363). DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer II following the manufacturer's protocols.