mare endometrium 25-28 day of pregnancy, biological replicate 3
Total RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, California, USA) according to the manufacturers protocol. RNA concentration and quality were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and a 2200 TapeStation instrument (Agilent, Santa Clara, USA); only samples with ratio A260nm/230 nm between 1.8 and 2.2 and RNA integrity number (RIN) values greater than 7.5 were used. MicroRNA libraries were prepared with the use of the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturers instructions. Specifically, following 3 adapter ligation, hybridization the Reverse Transcription Primer and 5 adapter ligation, Reverse Transcription and PCR amplification of obtained products were performed. The PCR was prepared with the use of 12 different indexed primers containing a 6 nt long unique sequence, which allows for barcoding the single library and multiplexing of the samples during sequencing. Next step included size selection (Novex 6% TBE PAGE gel, [Invitrogen, Carlsbad, California, USA]) of libraries. Subsequently, the quantity of obtained libraries were measured with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), while a 2200 TapeStation instrument (Agilent Technologies, Santa Clara, CA, USA) was used to assess their size. Afterwards, the obtained libraries were sequenced on a HiScanSQ sequencing instrument (Illumina, San Diego, CA, USA) according to the manufacturers protocol.