title: GSM4683872: IMX-101_10mg-L_RepB; Rana pipiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from whole tadpoles for all transcript expression experiments.  The same 5 individuals sampled per replicate for microbiome characterization were utilized in the RNAseq analysis for a total of 4 true replicates.  Each tadpole sample was individually flash frozen in liquid nitrogen and stored in a -80°C freezer.  RNA was extracted by homogenizing each individual tadpole in lysis buffer with a disposable pestle before RNA isolation with RNeasy mini kits (Qiagen, Valencia, CA). Total RNA concentrations were measured using a NanoDrop One Spectrophotometer (NanoDrop technologies, Wilmington, DE, USA). The integrity of total RNA for each tadpole was assessed on an Agilent 2100 Bioanalyzer (Palo Alto, CA).  Criteria for acceptable RNA integrity included a RNA integrity number > 7.0 from the Agilent 2100 Bioanalyzer and a 260/280 spectrophotometric reading > 2.0, a requirement enforced on each subsampled tadpole within the exposure replicate.  Tadpoles not meeting this requirement were not included in the pooled exposure replicate. RNAseq-based global transcriptomic expression assays were conducted for each of the munitions exposures.  The TNT exposure included control, 1.5, 2.3, and 3.4 mg/L exposures; IMX-101 included control, 10, 20, and 40 mg/L; NQ included control, 30, 150, 750 and 3750 mg/L exposures; and finally, MeNQ included control, 34, 170,  851, and 4225 mg/L exposures (nominal concentrations).  All RNA samples were shipped on dry ice to the US Army, Center for Environmental Health Research (USACEHR) for sequencing.  Once received, the total RNA samples were diluted, quantified using the Nanodrop 8000 spectrophotometer (Thermo Scientific, Waltham, MA), and 100ng total RNA was used to prepare libraries for sequencing using the TruSeq Stranded mRNA LT Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions.  Briefly, poly-A mRNA molecules were purified, fragmented, and copied into first strand cDNA.  Then the second strand cDNA was synthesized, the 3' ends were adenylated, single-index adapters were ligated onto the cDNA, and the products were enriched with PCR and purified to create the final cDNA library.  The quality of the libraries were determined using the D1000 ScreenTape on the Agilent TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA).  Libraries were then size selected using SPRIselect beads (Beckman Coulter Life Sciences, Indianapolis, IN) and quantified by the dsDNA HS Kit on the Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).  Next, the libraries were normalized, pooled (8 libraries per lane), and quantified using the dsDNA HS Kit on the Qubit 3.0 Fluorometer, followed by sequencing 1x150 cycles single-read on an Illumina HiSeq 4000 platform (Illumina) using the Illumina HiSeq 3000/4000 SBS kit following the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM4683872