GSM1570125: TG 1; Mus musculus; OTHER
Total intestinal epithelial cells from TRE-MSI2 mice treated with Dox for 24 hours were isolated as above, and wildtype intestinal crypts were isolated as above. For crosslinking, cell suspension was exposed to 2 pulses of 265nm UV light at 400mJ/cm2 in a Stratalinker (Model 2400, Stratagene). Epithelial cells were then lysed using PXL buffer (PBS, 01% SDS, 0.5% deoxycholate, 0.5% NP-40, plus protease inhibitor and RNAsin). The lysates were sequentially treated with DNaseI and RNase, and spin in ultra-microcentrifuge at 40,000g for 20 min. The supernatant was added to protein A Dynabeads (Dynal, 100.02) conjugated with MSI2 antibody (EMD Millipore 03-115) and incubated for 4 hours at 4 °C. 32P--ATP labeled 3’ RNA (RL-3) linker was ligated to the RNA fragment on beads overnight at 16°C. The beads were re-suspended in 30 ul of Novex loading buffer (without reducing agent), and separated with Novex NuPAGE 10% Bis-Tris gel and transferred to S&S BA-85 nitrocellulose membrane.  After overnight exposure, around 50 KD band was visualized and the corresponding membrane was cut to small pieces. The RNA was released by proteinase K digestion and isolated using RNA phenol and CHCl3 solution. 5’ RNA (RL-5) linker was ligated into the RNA fragments. The RNA was transcribed into complementary DNA using RT-PCR and amplified using Re-PCR with Solexa fusion primers.