title: GSM8371061: ptth,young-female,rep2; Drosophila melanogaster; RNA-Seq
library_construction_protocol: For RNA extraction. using Trizol method and following by DNase treatment(Ambion). RNA concentration was quantified by Qubit RNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA Catalog number: Q10210). RNA-Seq libraries were constructed using 300 ng of total RNA and NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs (NEB), Ipswich, MA, USA. Catalog number: E7420). RNA concentrations were measured using Qubit RNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA Catalog number: Q10210). Poly(A) mRNA was isolated using NEBNext Oligo d(T)25 beads and fragmented into 200 nt in size. After first strand and second strand cDNA synthesis, each cDNA library was ligated with a NEBNext adaptor and barcoded with an adaptor-specific index. Twelve libraries were pooled in equal concentrations, and sequenced using Illumina HiSeq 3000 platform (single-end, 50 bp reads format). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the cDNA was synthesized using random hexamer primers.  The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.