GSM4433224: HK017_MUT2; Danio rerio; RNA-Seq
Individual zebrafish embryos (5 d.p.f) from an incross of heterozygous carriers were homogenized in 100 μL of Tri Reagent (Sigma, Cat#93289) and transferred to 2 mL deep 96 well plates containing an additional 400μL of Tri Reagent. The RNA-containing aqueous phase was stored at -80°C until genotyping was completed. DNA was extracted from the interphase and organic phase by phenol-chloroform according to the manufacturer's instructions. Following genotyping, RNA was extracted from embryos homozygous mutants and sibling homozygous wt. DNA was removed from RNA extraction using TURBO DNA-free kit (Invitrogen, Cat#AM1907). RNA was quantified using Qubit RNA HS Assay Kit (ThermoScientific, Cat#Q32852) and the Qubit 4 Fluoromter (ThermoScientific, Q33226). RNA quality was checked by 18S/28S rRNA ratio using the Fragment Analyzer RNA Kit (ThermoScientific, Cat#DNF-471-0500) and the 5200 Fragment Analyzer System (ThermoScientific, Cat#M5310AA). cDNA libraries were prepared from 1μg of mRNA following poly-A selection using TruSeq stranded mRNA Library Prep (Illumina, Cat#20020595) according to manufacturer's instuctions. RNA-Seq was performed using mutant and wt sibling from each zebrafish line (n = 2 – 6). The libraries were sequenced in paired-end 75 bp mode at a depth of 25 million reads on an Illumina HiSeq 2500/3000 instrument.
GEO Accession
GSM4433224