GSM7901411: pSM33 cells, 10min 5EU feed; Mus musculus; RNA-Seq
Total RNA was harvested using RNeasy Minikit (QIAGEN). 5EU-labeled RNA was biotinylated by mixing samples with water, 100mM HEPES , 1 mM biotin picolyl azide (Click Chemistry Tools), Ribo Rnase Inhibitor, premixed 2mM CuSO4 and 10mM THPTA, and finally 12mM sodium ascorbate. Biotinylated RNA was then captured as follows: MyOne Streptavidin C1 Dynabeads (ThermoFisher Scientific) were first washed three times in Urea buffer (10mM HEPES, pH 7.5, 10mM EDTA, 0.5M LiCl, 0.5% Triton X-100, 0.2% SDS, 0.1% sodium deoxycholate, 2.5mM TCEP, 4M Urea) followed by three additional washes in M2 buffer (20mM Tris, pH 7.5, 50mM NaCl, 0.2% Triton X-100, 0.2% sodium deoxycholate, 0.2% NP-40). Washed beads were mixed with 3 parts 4M Urea buffer and 1 part biotinylated RNA and incubated for 60 min with 900rpm thermomixer shaking at room temperature. After magnetic separation, beads were washed 3 times with M2 buffer followed by 3 washes with Urea buffer at 37 C at 750rpm for 5 min. RNA was eluted from beads in 2 sequential elutions by incubating with elution buffer (5.7M guanidine thiocyanate, 1% N-lauroylsarcosine; both Sigma) at 65 C for 2 minutes, repeating with more elution buffer for a second elution. The elutions were pooled, diluted with Urea buffer, incubated with pre-washed streptavidin beads, washed, and eluted for 2 additional rounds exactly as described above for a total of 3 sequential captures. Final elutions were pooled, cleaned with Zymo RNA Clean and Concentrate following manufacturer's protocols (Banerjee et al., 2020). Captured RNA was used for library construction as previously described in Banerjee et al. 2020.