GSM3682869: control rep7; Bos taurus; RNA-Seq
Granulosa cells were thawed and resuspended in RLT buffer for RNA extraction using the RNeasy Micro kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Total RNA concentration was determined using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total RNA with a 28S to 18S ratio > 1 and RNA integrity number (RIN) ≥ 7 were used for RNAseq library construction. To produce RNAseq libraries, 200 ng of total RNA was used to isolate mRNA using NEBNext Poly(A) mRNA magnetic isolation module (New England Biolabs, Ipswich, MA, USA) and RNA library construction with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions. Briefly, extracted mRNA was fragmented in NEBNext first strand synthesis buffer by heating at 94°C, followed by first strand cDNA synthesis using reverse transcriptase and random primers. Synthesis of double-stranded cDNA was performed using the 2nd strand master mix provided with the kit. The resulting double-stranded cDNA was end-repaired, dA-tailing and ligated with NEBNext adaptors. Finally, libraries were enriched by 12 cycles of amplification and purified by Meg-Bind RxnPure Plus beads (Omega Biotek, Norcross, GA, USA).
GEO Accession
GSM3682869