Nanodrop nd800

We isolated genomic DNA using the ammonium acetate precipitation method [66 ]. We checked the integrity of the DNA using a 0.8% agarose gel stained with SYBRsafe (Invitrogen). We measured DNA concentration using either a fluorometer (FLUOstar OPTIMA) or Nanodrop ND800 (Thermo Fisher Scientific).

MicroRNA enriched total RNA was isolated from granulosa and theca samples using the miRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany), following the manufacturers protocol. Any genomic DNA contamination was removed by performing on-column DNA digestion using RNase-free DNase (Qiagen GmbH, Hilden, Germany). The quantity and purity of RNA was determined with NanoDrop ND-800 (Thermo Fisher Scientific, Wilmington, DE, USA) whereas the RNA integrity was evaluated using the RNA 6000 Nano Kit and 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). The granulosa RNA samples with the absorbance wavelength ratio (A260/A280) of ≥1.9 and the RNA integrity number of ≥7 were used for miRNAs deep sequencing. A total of 12 granulosa samples (three biological replicates of granulosa cells from SF or DF at day 3 or day 7 of the estrous cycle) were used for miRNA deep sequencing. However, the total RNA samples from theca cells were used for measuring the expression of candidate miRNAs generated from granulosa cells.

The quantity of LR-PCR DNA templates (prior and during library preparation) was assessed by spectrophotometry (Nanodrop ND-800, Thermo Scientific, Waltham, MA, USA), fluorometry (Qubit, Invitrogen, Carlsbad, NM, USA) and quantitative PCR (PicoGreen dosage with Quant-iT™ PicoGreen® dsDNA Assay Kit, Invitrogen on ABI 7900HT, Applied Biosystems). After quality controls, six libraries (one per individual) were prepared using the TruSeq DNA Sample Prep Kits v2 and TruSeq Universal Adapters (Illumina, San Diego, CA, USA). A TruSeq Universal Adapter was used for each DNA library in order to separate reads from different individuals after DNA sequencing. Library sizes were checked on BioAnalyzer chips (Agilent Technologies, Santa Clara, CA, USA). Paired-end library sequencing was carried out on the Illumina MiSeq plateform (2 × 250 bp chemistry) at the GeT-PlaGe lab (GenoToul, Toulouse, France).