title: GSM8127866: Delta-disordered (DMS-seq); Homo sapiens; OTHER
library_construction_protocol: HCT116 NORAD WT and NORAD–/– cells were seeded in a 6-well plate (200,000 cells per well). After 24 hr, NORAD–/– cells were transfected with NORAD constructs using PEI reagent. Cells were washed 48 hr post-transfection and incubated with warm DMEM containing 2% DMS for 5 min at 37°C. In parallel, as a control, 2% of DDW was added to another well of cells. Media with DMS was decanted, and cells were washed with 30% of beta-mercaptoethanol solution in PBS. The adherent cells were dislodged using a cell-scraper, and collected by centrifugation at 1000 × g at 4 °C for 5 min. Cells were resuspended in TRI reagent and RNA was isolated according to the manufacturer protocol. Total RNA was DNase-treated for 30 min at 37 °C in 1× TURBO DNase buffer with 1 μl TURBO DNase enzyme (Thermo Fisher Scientific). Reverse transcription was performed in a 10 μl volume with 1 pmol of gene specific primer (Table S7). To begin, a mixture of 3-6 µg of RNA with primer was incubated at 95°C for 30 sec to denature the template, and then it was returned to ice to anneal the primer to the template. To initiate the reaction, 20 U Marathon RT (company, cat#) with RT buffer (100 mM Tris-HCl pH 8.3, 400 mM KCl, 2 mM MnCl2, 10 mM DTT, 40% glycerol)  and 5 mM dNTPs were added to annealed substrate, and allowed the reaction to proceed for 1 h at 420C  (Guo et al. 2020; Mitchell et al. 2023).  Two ∼ 300 nt amplicons tiled around NRU 7 to 8 were designed to obtain sequencing coverage of the structured region of NORAD (Table S6). Amplicons had an overlapping of 65 nucleotides. Amplicons were generated using Q5 High-Fidelity DNA Polymerase (NEB) with NORAD specific forward and reverse primers, and 2 µl of cDNA per reaction carried out in a 25 µl total volume per amplicon.