GSM9476457: TTTRT1-2; Oryctolagus cuniculus; miRNA-Seq
Total RNA was extracted from 200 μL of rabbit peripheral blood plasma using the miRNeasy Serum/Plasma Kit (Qiagen, Germany) following the manufacturer's instructions. Briefly, plasma samples were mixed with QIAzol Lysis Reagent, followed by chloroform phase separation to isolate the aqueous phase containing RNA. The RNA was then bound to a silica membrane column, washed with RWT and RPE buffers, and eluted in 14 μL of RNase-free water. The concentration and integrity of small RNA were assessed using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA), respectively. Small RNA libraries were constructed using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, USA). Briefly, 1–10 ng of total RNA was ligated to 3′ and 5′ RNA adapters using T4 RNA ligase. Reverse transcription was performed to generate cDNA using M-MuLV Reverse Transcriptase, followed by PCR amplification (12 cycles) with index primers to introduce sample barcodes. The amplified libraries were size-selected (140–160 bp) using 6% polyacrylamide gel electrophoresis to enrich for miRNA-derived fragments. The final libraries were quantified using the Qubit dsDNA HS Assay Kit and validated for size distribution on the Agilent 2100 Bioanalyzer.