GSM8698501: RNAseq,shRPS29,rep1; Homo sapiens; RNA-Seq
For RNA-seq,total RNA was extracted using the RNAprep Pure Cell/Bacteria Kit (TIANGEN; DP430) following the manufacturer's protocol. For Ribo-seq, cells were treated with a specific lysis buffer containing cycloheximide (50 mg/mL) to obtain the lysate. The concentration of the lysate was measured using a Qubit fluorometer. To digest RNA other than ribosome-protected fragments (RPFs), cell or tissue lysates were treated with the unspecific endoribonuclease RNase I. Monosomes were isolated by size-exclusion chromatography and purification. Both ends of the RPFs were phosphorylated and ligated with 5' and 3' adapters, respectively. RNA samples were treated with an rRNA depletion kit (Qiagen; 334387) to minimize rRNA contamination. For RNA-seq, the extracted RNA was utilized for library construction with the Illumina TruSeq Stranded Total RNA Library Prep Kit. The library was sequenced using paired-end 150-bp reads. For Ribo-seq, the RNA fragments were reverse transcribed and amplified by PCR, followed by library construction using the Multiplex Small RNA Library Prep Set for Illumina (NEB; E7300L). The libraries were subjected to Illumina sequencing with single-end 50bp (SE50) sequencing.