GSM7728590: Spleen_ARV_rep2_Ribo; Gallus gallus; OTHER
Spleen tissue from each group was added to lysis buffer, ground at low temperature, and then low concentrations of RNase I were added for digestion. The digested samples were pooled and layered on the surface of 15 ml sucrose buffer (30% sucrose in RB buffer). The ribosomes were pelleted by ultracentrifugation at 42500 rpm for 5 h at 4 °C. RPF extraction was then performed using TRIzol, and ribosomal RNA (rRNA) was depleted using the Ribo-off® rRNA Depletion Kit (Vazyme) following the manufacturer's instructions. Sequencing libraries of RPFs were constructed following the VAHTS® Small RNA Library Prep Kit for Illumina (Vazyme). The library was resolved by a 6% polyacrylamide gel. The fraction with an insertion size of ~28 nt was excised and purified from the gel. This fraction was sequenced by an Illumina HiSeq-2000 sequencer for 50 cycles. High-quality reads that passed the Illumina quality filters were kept for sequence analysis.