JMM
Juvenile male mix
DNase I (RNase free) from Promega was used to eliminate genomic DNA contamination and 1 g DNase I-treated total RNA of each sample was used as the beginning material. The NEBNext? rRNA Depletion Kit was used for rRNA depletion. After that, the RNAs were purified by using the Agencourt RNAClean XP Beads from Beckman Coulter. Subsequently, RNA fragmentation, first strand and second strand cDNA synthesis and double-stranded cDNA end repair were performed using the NEBNext? Ultra? Directional RNA Library Prep Kit for Illumina?. Double strand cDNAs were purified using the Agencourt AMPure XP from Beckman Coulter and ligated to adaptors of the NEBNext Multiplex Oligos for Illumina. Finally, the Q5 Hot Start HiFi PCR Master Mix (NEB) was used for PCR enrichment of the adaptor-ligated DNA. Concentration and quality of the libraries were measured by using the Agilent High Sensitivity DNA Kit and a Bioanalyzer 2100 from Agilent Technologies.
JMM
SUB4273789
JMM