GSM4256979: RNA-seq_WT_3; Danio rerio; RNA-Seq
To perform RNA-seq on WT and thrb stl628/stl628 retina, we first isolated the retinas of 6-month-old WT and clutch-matched thrb stl628/stl628 zebrafish. Two retinas from a male and two retinas from a female zebrafish were combined to make one sample. Three WT and thrb stl628/stl628 samples were collected and RNA extracted using trizol-chloroform extraction. RNA concentrations ranged between 27-55 ng/μl, and all RNA integrity number scores were > 8.7. 2-4 μg mRNA were used for library preperation. First, Ribosomal RNA was removed by poly-A+ selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). Then, mRNA was fragmented in buffer containing 40 mM tris acetate pH 8.2, 100 mM potassium acetate, and 30 mM magnesium acetate heated to 94o C for 150 seconds. Subsequently, mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies), and a second-strand reaction was performed to produce cDNA. cDNA ends were made blunt, an “A” base was added to the 3' ends, and Illumina sequencing adapters were ligated to the ends. Ligated fragments were then amplified for 12 cycles using primers incorporating unique index tags.
GEO Accession
GSM4256979