GSM8489737: KC_45apf_pooled; Drosophila melanogaster; RNA-Seq
Cells were sorted into Trizol-LS and stored at -80C until RNA isolation. For RNA extraction, we followed the standard Trizol-LS protocol until the aqueous phase was isolated. We then passed the aqueous phase over Arcturus Picopure columns, including a DNAse treatment on the column. We quantified total RNA using a BioAnalyzer and aimed for 1ng starting material per library. As insect processing of rRNA makes the “RNA Integrity Number” irrelevant, we visually inspected total RNA profile to assess RNA quality. For each replicate, we matched the amount of total RNA used for library prep across the three cell types, i.e. we matched Kenyon cell and “double negative” RNA amounts to the amount we obtained for olfactory PNs. Total RNA was poly-A selected and libraries were prepared using SmartSeq V2 at the Rockefeller University Genomics Core and sequenced to a depth of 25 million reads using HiSeq 50 SR (adult samples) or NextSeq High Output 75SR (45h APF samples).