NextSeq 500 paired end sequencing; Drosophila melanogaster whole embryo RNA-Seq experiments during embryogenesis
Drosophila melanogaster whole embryo RNA-Seq experiments during embryogenesis
"Genotyped lines were selected from the 205 in the DGRP collection based on three criteria: 1) including lines with the highest quality and depth of genome sequencing, 2) avoiding pairs of highly-related lines and (3) avoiding lines with unusual levels of residual heterozygosity. Freshly enclosed adults were placed in embryo collection vials, with standard apple cap plates. After three/four 1 hr pre-lays, the flies were allowed to lay for 2 hrs, after which the embryos were aged to the appropriate time-point. The embryos were then dochorionated using 50% bleach, snap frozen in liquid Nitrogen and store at -80C." "~100 embryos were homogenized with Cordless Motor for Pellet Mix and pestels (VWR) in ice. RNA was extracted in TRIzol[tm]LS (Life Technologies), digested with RNAse free DNase I (Roche) and purified with the RNeasy mini kit (QIAGEN) according to the manufacturers' recommendations." Libraries were prepared following instructions of the NEBNext ultra directional RNA library prep kit for Illumina sequencing (NEB).