GSM8964784: 28, MCF10A, FBL, TREATED-FBL_siRNA, 15min 5eU pulse, 90 min chase; Homo sapiens; RNA-Seq
RNA isolation was performed with Qiagen RNeasy Mini kits followed by DNAse digestion (TURBO DNase, Invitrogen, AM2238) to remove genomic DNA. RNA concentrations were measured with Qubit RNA BR (Fisher Scientific, Q10211) and RNA integrity was analyzed on Agilent RNA High Sensitivity (HS) Tapestation. Total RNA (10-15 µg) isolated after 5eU-pulse chase was click-reacted with biotin picolyl azide (Click chemistry tools, 1167-25) as described in Bhat, P., et al., Nature. 2024 with the following modifications. Capture of biotinylated RNA was performed using 20uL Dynabeads MyOne Streptavidin C1 beads (Invitrogen, 65002) after click chemistry. Captures and washes were performed as previously described except for a modification to the 3 x 5 min washes of captured material, which were changed to 75° C in No Salt Urea buffer (4M Urea, 10mM HEPES, pH 7.5, 10mM EDTA, 0.5% Triton X-100, 0.2% SDS, 0.1% Na-DOC). We found that 3 rounds of sequential captures (as described by Bhat et al, 202432) as well an optimized protocol introduced here performing washes at high temperature and in a buffer lacking salt were essential to reduce background of highly abundant mature rRNA. Captured RNA was used for library construction as previously described in Banerjee, A., et al. Cell. 2020.