GSM2113454: Asexual Females rep1; Daphnia pulex; RNA-Seq
Collections of whole D. pulex individuals (~50-75) were homogenized using a small pestle in microcentrifuge tubes containing lysis buffer. Total RNA was isolated using solid phase extraction (Bioline, Inc.). Each sample was then snap-frozen in liquid nitrogen and stored at -80C. Library construction utilized the nAnT-iCAGE protocol (Murata et al., 2014). Briefly, first-strand reverse transcription was performed with total RNA from each sample using a random primer (N6 plus 3). The 5' m7G cap was oxidized (with sodium peroxide) and biotinylated using biotin (long arm) hydrazide. Biotinylated RNA/cDNA hybrid molecules were pulled down using magnetic streptavadin beads. After RNase-treatment of captured hybrids, a barcoded linker oligo was ligated to the 5' end of the cDNA molecules, follwed by ligation of a 3' linker. Second-strand synthesis was completed using primers that anneal to the 5' linker and anchor primer, creating the final dsDNA product.
GEO Accession
GSM2113454