GSM1319812: TG rep1; Mus musculus; OTHER
Total intestinal epithelial cells from two individual TRE-Msi1 mice treated with Dox for 24 hours were isolated as above, and wildtype intestinal crypts from two control mice were isolated as above. For crosslinking, cell suspension was exposed to 2 pulses of 265nm UV light at 400mJ/cm 2 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 spun in ultra-microcentrifuge at 40,000g for 20 min. The supernatant was added to protein A Dynabeads (Dynal, 100.02) conjugated with Msi1 antibody (AB5977, Millipore) 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 µl 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.