GSM9177149: J-Gammal cells, NUDT21 knockdown, replicate 1  [PAS-seq]; Homo sapiens; RNA-Seq
Total RNA was extracted using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, RC112-01). For each sample, 5 μg of total RNA was treated with RQ1 DNase (M6101,Promega, USA) to remove DNA, fragmented RNAs were used for PAS-seq library preparation. mRNAs were captured with mRNA Capture Beads kit （N401,Vazyme,Chian）. Fragmented mRNAs were used for directional PAS-seq library preparation by KAPA Stranded mRNA-Seq Kit for Illumina® Platforms (KK8544, Roche, USA). Fragmented mRNAs were converted into double strand cDNA. Following end repair and A tailing, the DNAs were ligated to Diluted Roche Adaptor (KK8726, Roche, USA). After purification of ligation product and size fractioning to 300-500 bp, the ligated products were amplified and purified, quantified and stored at -80℃ before sequencing. The strand marked with dUTP (the 2nd cDNA strand) is not amplified, allowing strand-specific sequencing. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina Novaseq Xplus system for 150 nt paired-end sequencing.