GSM8626287: E16.5 primary cortical cells, 8 hr, replicate C; Mus musculus; OTHER
Cells from a single well per replicate were lysed 0, 2, 4, 8, and 12 hr after addition of uridine by addition of RLT buffer (Qiagen RNeasy Plus Micro Kit, cat. 74034) supplemented with 1 mM DTT. Lysates were stored at -80 °C before further processing. RNA was extracted from lysates using the Qiagen RNeasy Plus Micro Kit according to manufacturer's instructions, except all buffers were supplemented with 1 mM DTT. Alkylation of 4sU RNA was performed as previously described (Herzog et al., Nat. Methods 2017) using ~1.5 µg of RNA per replicate. After alkylation, RNA was re-purified using the RNA Clean and Concentrator Kit (Zymo 11-325). Sequencing libraries were generated using 400 ng of alkylated input RNA per sample and the Illumina Stranded mRNA Prep Kit (cat. 20040532) according to the manufacturer's instructions.