title: GSM4290089: larvae, POP70_1; Danio rerio; RNA-Seq
library_construction_protocol: RNA from samples for RNA-seq analysis were purified using NucleoSpin® RNA extraction kit (Macherey-Nagel, Germany). RA1 lysismix (Macherey-Nagel, Germany) was added to each sample (10 larvae per sample) and samples were passed through a 21-gauge needle (HSW HENKE-JECT®, Germany) until complete homogenization. Total RNA was extracted from samples following manufacturer's instructions. Each sample was eluted in 50 μL RNase-free water and stored at -80 °C until further analysis A total of 2 μg was used for library preparation. A quality check (QC) of total RNA was performed with a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA), Agilent 2100 (Agilent Technologies, CA, USA) and agarose gel electrophoresis prior to library construction. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations. Briefly, mRNA was purified from total RNA using poly-T oligo attached magnetic beads. Fragments were randomly created in fragmentation buffer. First strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina, CA, USA) was added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand. The final cDNA library was ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection, and PCR enrichment. PCR products were purified (AMPure XP system, Beckman, US) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Libraries were analyzed with a Hiseq 4000 (Illumina, Ca, USA), using 150bp paired-end reads, with a depth of 20 million reads per sample.
attributes.tag: GEO Accession
attributes.value: GSM4290089