title: GSM2817250: dper_RE_m_r4; Drosophila persimilis; RNA-Seq
library_construction_protocol: Flies were anesthetized using CO2, and all samples were dissected in phosphate buffered saline solution (PH 7.4, Life Technologies, Carlsbad CA) and transferred to 56 or 112 μL of RNAlater (Life Technologies, Carlsbad CA) in 96-well 1.5 mL plates at room temperature after being macerated with forceps. Plates were stored at -80 oC at the NIH after dissection (melanogaster samples) or after dissection and shipping (non-melanogaster samples).  For RNA extraction, we added 20-30 1.0 mm glass beads (BioSpec Products, Bartlesville OK) to each sample and added RLT buffer from the RNeasy 96 kit (Qiagen, Valencia CA) to 712 μL (final volume).  We homogenized the samples three times for 60 seconds each with a mini-beadbeater (Biospec Products, Bartlesville OK). Extraction of total RNA was performed using the RNeasy 96 kit (spin version), except that we used 712 μL of 70% ethanol due to the increased volumes of RNAlater/RLT mixture.  RNA was quantified using Quant-iT RiboGreen (Life Technologies, Carlsbad CA).  We spot checked RNA integrity by capillary electrophoresis on a TapeStation 2200 (Agilent Technologies, Santa Clara CA) and found that the rRNA bands were distinct. We used 33 ng aliquots of RNA of three genetically distant species for each library, except for thorax, where we used 17 ng due to lower yield. The mixing of RNA was performed using a pipetting robot (Andrew Alliance, Vernier CH). RNA from D. melanogaster genitalia was not mixed with tissues of the other species. We used 100 ng of total RNA as input (except thorax samples: 50 ng), and followed the TruSeq PolyA+ stranded mRNA kit protocol with half volume reactions (Illumina, San Diego CA).  For each sample, we also added 10 pg Standard Reference Material 2374 beta (Pool 78A or 78B) ERCC spike-ins (Lee et al., Journal of Genomics, 2016, PMID 27512518) to the Fragment-Prime-Finish Mix during the mRNA fragmentation step.  We used 96-plex molecular barcoding (Kozarewa and Turner, Methods in Molecular Biology, 2011, PMID 21431778) from the Illumina TruSeq kit for all samples. Libraries were quantified with Quant-iT PicoGreen (Life Technologies, Carlsbad CA). For each sample, we conducted RNA-seq for the three libraries with the highest DNA concentration out of four libraries. The quality (i.e., no overt primer dimers or low complexity) of all selected libraries were confirmed by capillary electrophoresis on a TapeStation 2200 (Agilent Technologies, Santa Clara CA). We performed single-end 76 bp PolyA+ sequencing on the HiSeq2000 Sequencing System (Illumina, San Diego CA).
attributes.tag: GEO Accession
attributes.value: GSM2817250