title: GSM1402308: 2-cell embryos (polyA replicate 2); Caenorhabditis elegans; RNA-Seq
library_construction_protocol: Oocytes: Adult wild-type hermaphrodites (N2) were cut using a razorblade in PBS containing 0.5% BSA and 0.02%Tween. Oocytes were picked by mouth pipetting under a stereo microscope (Leica), washed thoroughly in PBS and lyzed in TriZol LS (Invitrogen, USA). Sperm: Sperm was isolated in principle as described previously(L’Hernault & Roberts, 1995) from males worms obtained from a fog-2(q71) mutant background strain. Males were cut in cold PBS containing protease inhibitors (Sigma-Aldrich, Germany). Sperm was subsequently purified by filtration (3x40µm, 2x10µm, 1x6µm nylon mesh) and washed twice in cold PBS. Sperm was then activated by incubation in PBS containing 200µg/mL Pronase (Sigma-Aldrich, Germany) for 30 minutes at 24°C. 1-cell embryos: 1-cell embryos were obtained by fluorescence activated cell sorting (eFACS) as described previously(Stoeckius et al, 2009) in an FACSAriaIII flow cytometer (BD Biosciences, USA). mRNA-Seq: PolyA RNA was fragmented into approximately 250 nt fragments by chemical fragmentation (200 mM tris acetate pH 8.2, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for exactly 3.5 min in a thermocycler. Fragmented RNAs were isolated with RNA Clean beads (Beckman Coulter, USA) according to manufacturer’s instructions. Fractionation was checked by capillary electophoresis in a RNA Pico 6000 chip using the Bioanalyzer (Agilent Technologies, USA). First strand cDNA synthesis was accomplished using Superscript III Reverse Transcriptase and random hexamers (Invitrogen, USA), followed by second strand synthesis using DNA Polymerase I and RNaseH (Invitrogen USA). Double-stranded DNA was purified with Agencourt AMPure beads XP (Beckman Coulter, USA) and quality was checked by capillary gelelectrophoresis on the Bioanalyzer with the Agilent DNA 1000 kit (Agilent Technologies, USA). dsDNA libraries subsequently processed for sequencing using the Genomic DNA Sample Prep Kit (Illumina, USA) according to the manufacturer’s protocol. For small RNA-seq: 1 µg of total RNA per sample were used for small RNA library preparation. First total RNA was treated with RNA 5’ polyphosphatase (Epicentre Biotechnologies, USA) prior to small RNA library preparation to allow for subsequent cloning of multiphosphorylated endo-siRNAs. Small RNA cloning and library preparation was then performed with the TruSeq Small RNA Sample Preparation Kit (Illumina, USA) according to the manufacturer protocol.
identifiers.namespace: GEO
identifiers.id: GSM1402308