GSM3108900: 10_CGATGT_L007_R1_001; Gasterosteus aculeatus; RNA-Seq
Individuals for brain gene expression profiling were gently netted directly from their home tanks and rapidly decapitated with sharp scissors. Heads were flash frozen and stored at -80°C until dissection. We first scraped the skull with rongeurs to expose brain tissue. Heads were placed in RNALater for 24 hours at 4°C. We then dissected whole brains in RNAlater (Thermo Fisher Scientific) on dry ice and extracted RNA using the PicoPure RNA Isolation Kit with optional DNase treatment (Thermo Fisher Scientific). Poly-A RNA was enriched from 1–2 μg of total RNA by using Dynabeads Oligo(dT)25 (Life Technologies), following the manufacturer's protocol. Two rounds of poly(A) enrichment were performed with a final elution in 14μL of water. The poly-A–enriched RNA was used to prepare RNAseq libraries, using the Illumina TruSeq kit (Illumina). Manufacturer's instructions were followed and 13–15 cycles of PCR amplification were performed depending on the starting input of total RNA. All samples were barcoded, libraries were quantified on a Qubit fluorometer using the dsDNA High Sensitivity Assay Kit (Life Technologies), and library size was assessed on a Bioanalyzer High Sensitivity DNA chip (Agilent). Libraries were pooled and diluted to a final concentration of 10 nM. Final library pools were quantified using real-time PCR, using the Illumina compatible kit and standards (KAPA) by the W. M. Keck Centre for Comparative and Functional Genomics at the Roy J. Carver Biotechnology Centre (University of Illinois). Single-end sequencing was performed on an Illumina HiSeq 2500 instrument by the W. M. Keck Centre for Comparative and Functional Genomics at the Roy J. Carver Biotechnology Centre (University of Illinois). Not all individuals yielded enough RNA, therefore the final sample sizes for RNA-seq informatics were no paternal cue/no personal cue: N = 5, paternal only: N = 9, personal only: N = 9, both: N = 7.
GEO Accession
GSM3108900