Computational protocol: Transformation of the intestinal epithelium by the MSI2 RNA binding protein

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Protocol publication

[…] Clip libraries were made as previously described in with minor modification. 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-γ-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. The CLIP library underwent single-end sequencing on an Illumina hiSeq2000 at the University of Pennsylvania Functional Genomics Core. RL-3: 5’-OH GUG UCA GUC ACU UCC AGC GG 3’ –puromycin; RL-5: 5’ –OH AGG GAG GAC GAU GCG G 3’-OH. Adapter sequences were removed from the 3' end using cutadapt (http://journal.embnet.org/index.php/embnetjournal/article/view/200) with options '-a GTGTCAGTCACTTCCAGCG -e 0.06 -O 6 -m 12'. Reads were then mapped to the mouse (mm9) transcriptome and genome using Tophat with options '--read-mismatches 1 --read-gap-length 1 --read-edit-dist 1 --max-multihits 100 --b2-very-sensitive --transcriptome-max-hits 100 --no-coverage-search --no-novel-juncs'. Peaks were called using Piranha with a window size of 200bp. Motifs were identified using the MEME software suite. […]

Pipeline specifications

Software tools cutadapt, TopHat, Piranha
Application CLIP-seq analysis
Organisms Mus musculus
Diseases Neoplasms, Colorectal Neoplasms