GSM7781203
Total RNA was extracted using a TRIzol™Reagent Kit (Invitrogen, Carlsbad, CA). RNA concentration and integrity were examined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany), and RNA samples with an RNA integrity number (RIN) > 8 were used for subsequent experiments. Nine small RNA libraries were established using the total RNA isolated from mammary gland tissue at the LL, DP and LG stages (LL1, LL2, LL3, DP1, DP2, DP3, LG1, LG2 and LG3) with an NEBNext®Ultra™RNA Library Prep Kit (Illumina, San Diego, USA). For each small RNA library, 1 μg of total RNA was subjected to electrophoresis using 12% Tris-borate ethylenediaminetetraacetic acid (TBE)-urea polyacrylamide gel electrophoresis (PAGE) gels (Invitrogen) to obtain small RNA fragments with a length between 18 and 30 nt. The 3' and 5' ends of the RNA were ligated with T4 RNA ligase 2 and reverse transcribed to obtain cDNA based on the 3' and 5' adapters ligated to the small RNA. The target library was amplified using PCR, and dsDNA fragments between 140 and 160 bp in length were extracted using TBE gel electrophoresis. The libraries were subjected to sequencing using an Illumina/Solexa system after quality tests.