GSM4232666: 28 hpf JAK1-A634D transgenic rep 2; Danio rerio; RNA-Seq
Each total RNA sample was extracted from 30-50 zebrafish embryos by homogenizing them in 500 µL Trizol reagent (Thermo Fisher Scientific, 15596026) using 1 mL syringe and 22G needle. Lysates were centrifuged at 12,000 g at 4ºC for 5 minutes and the supernatant was transferred to Phasemaker Tubes (Thermo Fisher Scientific, A33248) and RNA was purified according to the Phasemaker Tubes protocol manual. Total RNA samples were measured and analyzed for integrity on Agilent Tape Station. Samples with RNA integration number ≥8 were selected for library preparation. Poly-A enrichment was performed from 20 µg of total RNA to enrich for mRNA (Dyna beads mRNA direct micro Kit). 100ng of poly-A enriched RNA was fragmented using RNase III and purified using magnetic bead clean up module (RNA Seq V2 kit, Life Technologies). The size distribution of the fragmented RNA was assessed on Agilent Tape Station using RNA HS screen tape assay and 50 ng of fragmented polyA-enriched RNA was used to prepare whole transcriptome library (RNA seq V2). Yield and size distribution of the library was analyzed on Agilent Tape Station using D1000 screen tape. Barcoded library was equally pooled and amplified onto Ion Sphere™ Particles (ISPs) from Ion Pi HiQ OT2 kit (Life Technologies). ISPs enriched with template library were loaded onto Ion PI chip V3 and sequenced on Ion Proton from Thermo Fisher.
GEO Accession
GSM4232666