fRIP EED Control Replicate 3
C2C12 cells were collected by spinning for 5 minutes at 500 RCF, followed by washing using room temperature 1XPBS (phosphate-buffered saline). Each 5x106 cells were resuspended in 1ml of media without FBS or HS. We then added formaldehyde (Sigma) of a final concentration of 0.1%. Cross-linking was done for 10 minutes at room temperature. Quenching were performed using glycine adding to a final concentration of 125mM. The cells were spun again for 5 minutes at 500 RCF, then washed twice using cold PBS. The frozen pellets were then resuspended in RIPA lysis buffer (50mM Tris (pH 8.0), 150mM KCl, 0.1% SDS, 1% Triton X-100, 5 mM EDTA and 0.5% sodium deoxycholate, supplemented with freshly prepared 0.5 mM DTT, 1X EDTA protease inhibitor cocktail (Roche) and 100 U/ml RNaseOUT (Life Technologies, 10777019) per tube.The cells were incubated at 4C for 10 minutes, then lysed with a Branson digital sonicator using 10% amplitude for 0.7 seconds ON and 1.3 seconds OFF at 30-second intervals for a total of 90 seconds. Lysate was then centrifuged at maximum speed for 10 minutes at 4C. Supernatant was collected and an equal amount of f-RIP binding/wash buffer (150mM KCl, 25mM Tris (pH 7.5), 5mM EDTA, 0.5% NP-40, supplemented with freshly prepared 0.5 mM DTT, 1X EDTA protease inhibitor cocktail (Roche) and 100 U/ml RNaseOUT (Life Technologies, 10777019)) was added to dilute the samples. To start the f-RIP, the lysates were thawed and 5g of EED (Millipore, 03-196 | RIPAb+TM EED) or IgG (Santa Cruz, sc-2027) was added. Then the lysates plus the antibody were left to rotate at 4C overnight before adding the Dynabeads Protein G. The Dynabeads were added at a concentration of 50l per 1ml of lysate for one hour, to recover immunoprecipitated complexes. Washes were carried out twice using the 1mL of f-RIP binding/washing buffer. After the final wash, the beads were removed and stored at -20C. Frozen beads were resuspended in 56l of RNase-free water, 33L of 3reverse-crosslinking buffer (3 PBS (without Mg or Ca), 6% N-lauroyl sarco-sine, 30mM EDTA, 15mM DTT (add fresh)), 10l of Proteinase K (Life Technologies, catalogue #AM9516). 1l of RNaseOUT was added to re-suspended the beads and input samples. Protein degradation and reverse crosslinking was done by incubating the samples and input for one hour at 42C, followed by a second hour at 55C. 1ml of Trizol (Sigma) was then added to the samples. Shaking the samples followed by adding 200l of chloroform, then vigorous shaking for ~15 seconds. RNA was precipitated and cleaned with Qiagen RNeasy min-elute column (Qiagen, catalog #74204).We performed library construction using Illumina Truseq stranded total RNA (Cat no. RS-122-2201) per the vendors instructions. Final library was pooled and sequenced at Illumina HiSeq 4000 platform.