GSM4159123: Ileum_ip_1; Mus musculus; RIP-Seq
For intestine immunoprecipitation (IP) mice were sacrificed and tissue remove and divided as above. Samples were washed of fecal contents in PBS with cycloheximide (0.2 mg/mL) (PBS/CHX).  Mesenteric fat was removed and the muscularis was separated from the mucosa as described above and samples were washed 5 times in PBS/CHX. For nodose tissues were isolated as described above. The RiboTag immunoprecipitation protocol (http://depts.washington.edu/mcklab/RiboTagIPprotocol2014.pdf)  was then followed with the following modifications: All samples were homogenized by hand with a dounce homogenizer in 2.5 mL supplemented homogenization buffer (changes per 2.5 mL: 50 µl Protease Inhibitor, 75 µl heparin (100 mg/mL stock), 25 µl SUPERase In RNase Inhibitor). Samples were then centrifuged for 10 minutes at 10,000 G, after which 800 µl of supernatant was removed and 5µL of anti-HA antibody (Abcam, ab9110) was added. For input samples, 40uL of supernatant was removed and 60uL of PicoPure lysis buffer was added. These input samples were then put at -80°C. Immunoprecipitated samples were kept rotating at 4°C with antibody for 1 hour. 200 µl of Thermo Protein magnetic A/G beads were washed with homogenization buffer, added to the sample, and kept rotating for 30 minutes at 4°C. The beads were washed four times with high-salt buffer and samples were eluted with 100 µl of PicoPure lysis buffer.  RNA was extracted using the Arcturus PicoPure RNA isolation kit (Applied Biosystems) according to the manufacturer's instructions.
GEO Accession
GSM4159123