title: GSM5007886: Asyn DMSO Rep C; Mus musculus; OTHER
library_construction_protocol: 2 million cells were lysed, washed, and fractionated by centrifugation to obtain nuclei. Nuclei were stored in glycerol freezing buffer until library preparation. PRO-seq libraries were prepared using biotin-NTP-supplemented run-ons from 0.2-0.5 x 10^6 permeabilized cells with Drosophila nuclei spike-in. NRO-RNA was purified using Norgen RNA isolation columns (Norgen Biotek cat. # 17200), followed by base hydrolysis on ice for 10min.  After the first binding and 3'-ligation, the NRO-RNA was bound to beads and all subsequent reactions (decapping, end repair, 5'-ligation, reverse transcription) were performed as in (Mahat et al., 2016), except that the reactions were done on the beads in 20μl. All biochemical steps after the 3'-ligation were performed with rotation except for reverse transcription. After reverse transcription, cDNA was eluted twice with 25μl ddH2O by heating for 1min at 80˚C, returning tubes to magnet, and collecting supernatant.