RNA-seq of Homo sapiens:Human colon tissue, Isolation OF CELL line
Using the structural feature that most eukaryotic mRNAs have polyA tails, mRNAs with polyA tails were enriched by Oligo(dT) magnetic beads, and then the mRNAs were randomly interrupted using divalent cations, and the fragmented mRNAs were used as templates and random oligonucleotides as primers for synthesizing cDNA first strand using the 1st Strand Enzyme reverse transcriptase system. The first strand of cDNA was synthesized in the 1st Strand Enzyme reverse transcriptase system using fragmented mRNA as template and random oligonucleotides as primers. The purified double-stranded cDNA was end-repaired, A-tailed and ligated to the sequencing junction, and the 200-300bp cDNA was selected for PCR amplification and the PCR product was purified again to obtain the final library. After the library was constructed, it was quantified using a Qubit 2.0 Fluorometer. After passing the library test, the library samples were sequenced by Illumina on an Illumina NovaSeq 6000 sequencing instrument in PE150 sequencing mode.