GSM3611162: DKK1_C; Bos taurus; RNA-Seq
RNA was extracted from each embryo using the Qiagen RNeasy Mini kit (Qiagen, Valenciam, CA, USA) following manufacturer instructions, including DNase treatment. 25 ng of total RNA was used for mRNA isolation with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). RNA library construction was performed at the Interdisciplinary Center for Biotechnology Research (ICBR) Gene Expression & Genotyping Core, University of Florida. RNA libraries were constructed with NEBNext® Ultra™ Directional RNA Library Prep Kit (New England Biolabs) according to the manufacturer's user guide. Briefly, RNA was fragmented followed by first strand cDNA synthesis using reverse transcriptase and oligo-dT primers. Synthesis of double strand cDNA was performed, followed by end-repair and adaptor ligation. At this point, Illumina adaptors were ligated to the sample. The library was enriched (each library had a unique barcode) by 10 cycles of amplification, and purified by Agencourt® AMPure beads (Beckman Coulter Life Sciences, Indianapolis, IN, USA). Finally, equimolar amounts of 20 individual libraries were pooled and sequenced by Illumina HiSeq 3000 2X100 cycles run for total of 3 runs (Illumina Inc., San Diego, CA, USA).
GEO Accession
GSM3611162