GSM6602006: SH-SY5Y_0.5h MPP+_Rep2; Homo sapiens; OTHER
SH-SY5Y cell nuclei were isolated with buffer A (10 mM Tris–HCl pH 7.5, 300 mM sucrose, 3 mM CaCl2, 2 mM MgAc2, 0.1% Triton X-100, 0.5 mM DTT) and quick frozen and stored at -80°C in buffer D (10 mM Tris–HCl pH 8.0, 25% glycerol, 5 mM MgAc2, 0.1 mM EDTA, 5 mM DTT) as described in Mahat et al. 2016. Nulcear run-on reaction was performed, Biotin RNA was fragmented in 0.2N NaOH, 3'RNA adapters (5'p-GAUCGUCGGACUGUAGAACUCUGAAC-/3InvertedT/) were ligated, 5'RNA adapters (5'-CCUUGGCACCCGAGAAUUCCA-3') were ligated to the 5' repaired RNA (RppH and PNK treated). Adaptor ligated nascent RNA was reverse transcribed using RP1 primer (5'-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3'). Final library was amplified using RP1 and barcoded RPIx (5#-CAAGCAGAAGACGGCATACGAGAT xxxxxx GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3') primers (As decribed in Mahat et al., 2016).