GSM2598409: Nmbu-RNA-12; Danio rerio; RNA-Seq
Total RNA was isolated with TRIzol Reagent (Invitrogen) according to manufactures’ instructions. Briefly, 1 ml TRIzol was added to each sample consisting of 100 embryos and homogenized using Magnalyser Beads (Roche Diagnostics). Each sample was eluted in 40 µl RNase-free water and stored at − 80°C until required. RNA purity and yield was determined using NanoDrop-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE) and RNA integrity number (RIN) was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) using RNA Nano LabChip Kit (Agilent Technologies), which were all of sufficient quality for sequencing (RIN > 9.0). One sample (control 0 yr) got lost during the RNA extraction and we proceeded with duplicate samples of the controls of 0 yr. Per sample, a total of 1.5 µg total RNA was used for library preparation. Non-directional libraries were generated using the NEBNExt Ultra mRNA kit (New England Biolabs, USA) according the manufacturers recommendation’s. mRNA was enriched using oligo (dT) beads, followed by fragmentation, followed by first strand cDNA synthesis using random hexamers and M-MuLV reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand. The final cDNA library is generated after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment. PCR products were purified with the AMPure XP system (Beckman, US) and library quality was checked on the Agilent Bioanalyzer 2100 system (Agilent, US). Libraries were analyzed in paired end 150 bp on the Hiseq 4000 (Illumina, US), using 150 bp paired end reads.
GEO Accession
GSM2598409