GSM8682918: TE1_NC-P; Homo sapiens; OTHER
Total RNA extraction using TRIZOL (15596-018, Ambion,USA). The RNA was further purified with two phenol-chloroform treatments and then treated with RQ1 DNase (M6101, Promega, Madison, WI, USA) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260nm/280nm(A260/A280) using Ultrafine spectrophotometer (N50 touch, IMPLEN, Germany).The integrity of RNA was further verified by 1.0% agarose gel electrophoresis. For each sample, 5 μg of total RNA was treated with RQ1 DNase (M6101, Promega) to remove DNA, fragmented RNAs were used for PAS-seq library preparation. mRNAs were captured with mRNA Capture Beads kit (N401, Vazyme). Fragmented mRNAs were used for directional PAS-seq library preparation by KAPA Stranded mRNA-Seq Kit for Illumina® Platforms (KK8544, Roche). Fragmented mRNAs were converted into double-strand cDNA. Following end repair and A tailing, the DNAs were ligated to Diluted Roche Adaptor (KK8726, Roche). After purification of the 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 the Illumina Novaseq Xplus system for 150 nt paired-end sequencing.