GSM5777057: ssDRIP_siNT_E2_rep1; Homo sapiens; OTHER
DRIP was performed on T-47D cells using an optimised method previously reported by Halász et al (PMID: 28341774). The best-performing protocol number 5 was adopted. In this experiment, S9.6 antibody from Kerafast (ENH002) was used. To enable strand-specificity, DRIP product was subjected to second-strand DNA synthesis with uracil incorporation. More specifically, DNA:RNA hybrid was reconstituted in 1× NEB first-strand synthesis buffer (NEB E7525) without the addition of primer and enzyme to a final volume of 20 µl. The product was immediately used as the starting material of NEB's directional second-strand synthesis method following manufacturer's instructions (NEB E7550). The resultant DNA was purified using SPRI beads (Thermo Fisher) at a volume ratio of 2.0 and eluted using TE buffer. After quality inspections by BioAnalyzer 2100 (Agilent), the product was sent to BGI, Hong Kong, where strand-specific libraries were constructed using DNB technology.