title: GSM5098882: Drosophila Pex5 knockdown samples, rep3; Drosophila melanogaster; RNA-Seq
library_construction_protocol: For Drosophila oenocytes, adult tissues oenocytes (20 females per replicate) were all dissected in cold 1 × PBS before RNA extraction. For oenocyte dissection, we first removed FB through liposuction and then detached oenocytes from the cuticle using a small glass needle. Tissue lysis, RNA extraction was performed using RNeasy Micro kit from QIAGEN (catalog number 74034) with the following modifications. Samples were first collected in 1.7ml centrifuge tubes. Dissected tissues were saved in 150ul of Buffer RLT (a component of RNeasy kits) with 143mM β-mercaptoethanol on ice during the dissection. Samples were then put at room temperature for 3 minutes prior to lysis and centrifuged at 7.5xg for 3minutes. Additional 150ul buffer RLT was added and pellet pestle grinder (Kimble pellet pestles, catalog number 749540-0000) was used to lyse the tissue for 1 minutes. 20ng carrier RNA was added to the cell lysates. Frozen samples was thawed in 37°C water bath for 1 minute. Total RNA was extracted using RNeasy Plus Micro columns following company manual. For human cell experiments, total RNA was extracted using TRIZOL. Fro Drosophila oenocytes, RNA-seq libraries were constructed using 100 ng of total RNA and NEBNext Ultra II RNA Lib Prep (NEB, Ipswich, MA, USA. Catalog number: E7770L). RNA concentrations were measured using Qubit RNA BR Assay Kit (Thermo Fisher Scientific, catalog number: 10210). Poly(A) mRNA was isolated using NEBNext Oligo d(T)25 beads and fragmented into 200 nt in size. Purification of the ligation products are performed using Beckman Coulter AMPURE XP (BECKMAN COULTER, catalog number: A63880). After cDNA synthesis, each cDNA library was ligated with a NEBNext adaptor and barcoded with an adaptor-specific index (NEBNext® Multiplex Oligos for Illumina, NEB, catalog number: E7335S). Twelve libraries were pooled in equal concentration and sequenced using Illumina HiSeq 3000 platform (single end, 150bp reads format). For human cells, RNA-seq libraries were constructed using 1ug DNA-free total RNA with NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA. NEB#E7770).  Poly (A) mRNA was isolated using NEBNext Poly(A) mRNA Magnetic isolation module (New England Biolabs, Ipswich, MA, USA. NEB#E7490). After first strand and second strand and second strand cDNA synthesis, each cDNA library was ligated with a NEBNext adaptor and barcoded with an adaptor-specific index. Twelve libraries were pooled in equal concentrations and sequenced using Illumina HiSeq 3000 platform.