title: GSM6919235: dsGFP_chromatin_associated_(>500bp_fraction) S17_C1L; Drosophila melanogaster; RNA-Seq
library_construction_protocol: Total RNA was extracted using Trizol (Ambion) and ethanol precipitated following standard procedures. For chromatin-associated RNA extraction,S2 cells were crosslinked with formaldehyde for 10 min at RT and the cross-linking was stopped by addition of 1 M glycine for 10 additional min. RNA was isolated with Trizol (Ambion) The cells were then collected and resuspended in Buffer 1 (50 mM Hepes pH 7.6, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0,25% Triton X100 and cOmplete protease inhibitors) and incubated for 10' at 4 °C, followed by centrifugation at 4 °C and 500g. The pellet was resuspended in cold Buffer 2 (200 mM Nacl, 1 mM EDTA, 0,5 mM EGTA, 10 mM Tris pH 8 and cOmplete protease inhibitors) and centrifuged again. Finally, chromatin was resuspended in Buffer 3 (1 mM EDTA, 0,5 mM EGTA and 10 mM Tris pH 8 and cOmplete protease inhibitors). Chromatin was fragmented using a Bioruptor sonicator (Diagenode) with 25 high intensity sonication 30'' on/off pulses. RNA was isolated with Trizol (Ambion) RNA-seq experiments consisted of biological triplicates, but the total RNA samples were pooled and sequenced together. Before RNA-sequencing, and in order to obtain adequate sequencing coverage for RNAs over such a broad size range, the RNA preparations were fractionated using AMPure XP paramagnetic beads (Agilent technologies) at a concentration of 0,6X to select RNAs larger than 500 nt and 1,6X to select caRNAs smaller than 500 nt. The 0-500 nt caRNAs were directly submitted to ribosomal depletion and library preparation, whereas caRNAs >500 nt were first fragmented by sonication. The NEBNext rRNA Depletion kit was used for ribosomal RNA depletion. Single-end strand-specific libraries were prepared using NEBNext Ultra RNA library Prep Kit for Illumina (NEB) according to the manufacturer's instructions. Samples were sequenced in a Illumina NextSeq 500 sequencer aiming for a depth of 15 million reads in average per sample with a length of 80 nucleotides per read