GSM2849538: Acf_PB_5; Drosophila melanogaster; RNA-Seq
Prior to RNA-seq, the AcfC flies were backrossed with the wild-type OrR strain for 8 generations. Embryos were collected 0-45 min AEL and allowed to develop at 25°C until approximately 30 min before the desired stage (around 1 h for Bownes Stage 3 and 4 h for Bownes Stage 8). Without prior dechorionation, embryos were hand-picked and submerged into a drop of Voltalef 10s halocarbon oil (Lehman and Voss Co.) placed on a microscope slide. After about 5 min, the embryonic structures become visible under the stereomicroscope. Embryos were allowed to develop further under the halocarbon oil until the desired stage. Single embryos were picked and crushed with a 26G needle into 200 µl of Lysis Buffer (supplemented with Proteinase K) from the Agencourt RNAdvance Tissue Kit (Beckman Coulter, Cat No A32645). After the addition of 10 µl of 1:100 ERCC Spike-in RNA mix (Ambion, Cat No 4456740), the samples were incubated at 37°C for 20 min, snap-frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted from the single-embryo homogenate using the same Agencourt RNAdvance Tissue Kit, following standard protocol but utilizing half of the volumes recommended. The RNA integrity was checked on a Bioanalyzer 2100 (Agilent). Ribosomal RNA depletion was achieved using rRNA Depletion Kit (Human/Mouse/Rat) (New Englands Biolab, Cat No E6310) and the rRNA-depleted RNA was stored at -80°C until further processing. Non-directional libraries were prepared using NEBnext Ultra RNA Library Prep Kit for Illumina (New Englands Biolab, Cat No E7530S) following standard protocol. 6 replicates per genotype and stage were sequenced on an Illumina HiSeq1500 instrument.