title: GSM4725469: colon_Snap25_cre-pos_ip_SPF_2; Mus musculus; OTHER
library_construction_protocol: Duodenum, ileum, and colon myenteric plexus was dissected into PBS with cycloheximide (0.2 mg/mL) (PBS/CHX). 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.