title: GSM3170908: dgriG1_f_ge_R3.ERCC; synthetic construct; RNA-Seq
library_construction_protocol: Flies were anesthetized by briefly chilling on a 0oC glass plate. All samples were dissected in Phosphate Buffered Saline solution pH7.4 and transferred to 112ul of RNA later (Life Technologies, Carlsbad, CA) in 96-well plates at room temperature after being macerated with forceps. After transport, plates were stored at -80oC. For D. grimshawi samples, we added 20-30 1.0mm glass beads (BioSpec Products, Bartlesville, OK) to each sample and added 600ul RLT buffer from the RNeasy 96 kit (Qiagen, Valencia, CA) to each well. 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 according to manufacturer's guide (spin version), except that we used 712μl of 70% ethanol in each well to dilute the RNAlater and RLT buffer. D. grimshawi RNA was added to D. melanogaster RNA and A. stephensi embryos for homogenization and a second round of RNA extraction using the RNeasy kit. D. melanogaster and A. stephensi RNAseq will be reported elsewhere. Four head and gonad samples of Wm1041 were split into mixed-species and single-species RNA preparations. All other samples were prepared without mixing with other species using the same RNeasy protocol. RNA was quantified using the Quant-iT RiboGreen quantification kit Life Technologies, Carlsbad, CA). We used the Truseq PolyA+ and stranded mRNA kit with half volumes (Illumina, San Diego, CA). 100ng total RNA was input for all samples. We also added 20pg Pool 78A or 78B ERCC spike-ins (Jiang et al., Genome Research, 2011, PMID 21816910) added to the Fragment-Prime-Finish Mix during mRNA fragmentation step. Libraries were quantified with Quant-iT PicoGreen (Life Technologies, Carlsbad, CA), and pooled for multiplexed sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3170908