Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: RNA was extracted from Drosophila 3rd instar larvae eye discs using a Qiagen kit followed by DNAse treatment. For smRNA-seq RNA was extracted from Drosophila 3rd instar eye discs using Qiagen kit with Trizol, spike-in controls added at the beginning of the protocol, and DNAse treatment was added at the end. For crosslinked ChIP, cells were crosslinked (1% formaldehyde, 10mins) and sonicated. Protein:DNA complexes were immunoprecipitated with specific antibodies pre-bound to protein A or goat anti-mouse IgG magnetic beads. Eluted DNA underwent crosslink reversal, and were purified using Qiagen PCR purification columns. For RNA extraction, 1 million TEX cells were pelleted, washed with PBS and resuspended in 350ul RLT buffer (Qiagen-RNAeasy mini kit) with beta-mercaptoethanol. The suspension was then stored at -80oC until frozen. Sample was thawed and RNA extraction was performed using Qiagen's RNAeasy mini kit as per the manufacturer's instructions. total RNA samples submitted to Genome Quebec facility for quality control assessment and library preparation. Illumina HiSeq 4000 with miRNA size select has been used for smRNAseq and Illumina HiSeq 4000 using mRNA stranded library has been used for RNA-seq. The eluted ChIP DNA undewent adaptor ligation based library construction for single-end 50bp Illumina sequencing on HiSeq 4000. RNA-seq library preparation was performed with ribosomal RNA (rRNA) depletion according to instructions from the manufacturer (Epicentre) to achieve greater coverage of mRNA and other long non-coding transcripts, paired-end sequencing (100 bp) was performed on the Illumina HiSeq 2500 or 4000 platform.