title: GSM7509750: Whole intestine epithelial cells, dsRNA (K1) IP, control replicate 1; Mus musculus; RIP-Seq
library_construction_protocol: Primary whole epithelial cells were isolated from the proximal half of the small intestine of adult mice (2 days after completing tamoxifen treatment) following incubation in EDTA for 50 minutes total at 4 C in tubes coated with 0.1% BSA in PBS. Isolated primary intestinal epithelial cells were resuspended in dsRIP buffer containing mammalian protease inhibitor and SUPERase RNase inhibitor for 5 minutes as previously described (Gao et al., 2021). After spin down, the supernatant was incubated with dsRNA J2 antibody (Cell Signaling Technology #76651) or dsRNA K1 antibody (Cell Signaling  Technology #28764) for 2 hours before adding to Protein G DynaBeads for 1 hour. RNA extraction was performed using Trizol and chloroform-based methodology, with two rounds of chloroform incubations to reduce phenol contamination. For pulldowns, RNA was extracted using an miRNeasy micro kit (Qiagen). Library preparation included random hexamers and strand-specific rRNA depletion with UMIs (2x150 bp).