GSM8844652: HEP3B, Control, Rep 1; Homo sapiens; OTHER
Total RNA isolation was performed using Trizol Reagent (ThermoFisher Scientific) according to the manufacturer's protocol. Total RNA was then DNase treated with DNase I (NEB) and reconstituted in nuclease-free water. Total RNA was dephosphorylated followed by a 3' adapter ligation. The 3' adapter contains a spacer that impedes the self-ligation of RNA. The ligated RNA mixture was 5' re-phosphorylated followed by ligation of a 5' RNA adaptor containing a Unique Molecular Identifier (UMI) of ten randomized nucleotides. Ligated RNA was then fragmented to obtain fragments of ~200 nt from the 5'end of the molecule. Fragmented RNA was ligated to another 3' RNA adapter. Final libraries were PCR amplified using Q5® High-Fidelity 2X Master Mix (NEB) and NEBNext® Multiplex Oligos for Illumina®. The primers aligned perfectly to the 5' adapter and the second 3' adapter. Each sample was assigned a different indexed primer.