title: GSM3672818: embryo20-21h_E3_G6; Drosophila melanogaster; RNA-Seq
library_construction_protocol: For RNA extraction, tubes were thawed on ice, supplemented with 350 µL of 100% Ethanol before homogenizing again with the same parameters. We then used the Direct-zol™ RNA Miniprep R2056 Kit, with the following modifications: we did not perform DNAse I treatment, we added another 2 min centrifugation into an empty column after the RNA Wash step, finally elution was performed by adding 8 µL of RNAse-free water to the column, incubation at room temperature for 2 min and then centrifugation for 2 min. RNA was transferred to a low-binding 96 well plate and stored at -80°C. RNA quantity was assessed using picogreen (Invitrogen P11496). Samples were then grouped according to their concentration in 96-well plates, and diluted to a final concentration determined by the lowest sample concentration on the plate. RNA was then used for gene expression profiling using BRB-seq. In short, the BRB-seq protocol starts with oligo-dT barcoding, without TSO for the first-strand synthesis (reverse transcription), performed on each sample separately. Then all samples are pooled together, after which the second-strand is synthesized using DNA PolII Nick translation. The sequencing library is then prepared using cDNA augmented by an in-house produced Tn5 transposase preloaded with the same adapters (Tn5-B/B), and further enriched by limited-cycle PCR with Illumina compatible adapters. Libraries are then size-selected (200 - 1000 bp), profiled using High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, #DNF-474), and measured using Qubit dsDNA HS Assay Kit (Invitrogen, #Q32851).
attributes.tag: GEO Accession
attributes.value: GSM3672818