GSM3933439: CRAC_Skiv2l_Focad_KO_4B_1; Mus musculus; OTHER
mESCs were grown in 2x 15-cm dishes to ~80 % confluency, dishes washed 2x with PBS, the PBS removed, then cells crosslinked on ice (with dishes facing up) in a Stratagene Stratalinker 2400 (400 mJ cm−2). Cells were lysed by incubating with 5 ml of either TN150+NP40 (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 0.5 % Nonidet P40 substitute and 1x cOmplete Protease Inhibitor Cocktail), or of RIPA (50 mM Tris-HCl pH 7.8, 300 mM NaCl, 1.0 % Nonidet P40 substitute, 0.1 % SDS, 10 % (v/v) glycerol, 0.5 % sodium deoxycholate, 1 mM beta-mercaptoethanol, 1x cOmplete Protease Inhibitor Cocktail), as indicated. The harsher buffer (RIPA) was initially used to ensure complete extraction, but as this can reduce FLAG binding we later switched to a milder buffer (TN150), which did not affect library content. The cells were further disrupted using a cell scraper then lysates collected and centrifuged (6500 xg for 20 min at 4 °C). Supernatants were frozen in liquid nitrogen and stored at -80 °C. Lysates were thawed on ice and incubated with 100 µL anti-FLAG M2 magnetic beads overnight. The supernatant was discarded and beads washed 3x with 1 ml TN150 (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 0.1 % Nonidet P40 substitute). Protein:RNA complexes were eluted by incubating beads in 1.5 ml TN150 supplemented with 5 mM beta-mercaptoethanol and 0.3 mg/ml 3xFLAG peptide, rotating at 4 °C for 2 hr. The eluate was then incubated with 50 µL Dynabeads M-280 Streptavidin, rotating at 4 °C overnight. Beads were washed 2x in TN600 (50 mM Tris-HCl pH 7.8, 600 mM NaCl, 0.1 % Nonidet P40 substitute, 5 mM beta-mercaptoethanol) and 2x in TN150 supplemented with 5 mM beta-mercaptoethanol. RNA was fragmented by incubating beads in 500 μL TN150 supplemented with 5 mM beta-mercaptoethanol and 1 μL of 0.1 U diluted RNace-IT. After 4 min at 37 °C, the RNases were denatured by replacing the solution with 400 μL WBI (50 mM Tris-HCl pH 7.8, 300 mM NaCl, 0.1 % Nonidet P40 substitute, 5 mM beta-mercaptoethanol and 4.0 M guanidine hydrochloride). The beads were washed 2x in WBI, then 3x in 400 μL 1xPNK (50 mM Tris-HCl pH 7.8, 10 mM MgCl2, 0.5 % Nonidet P40 substitute, 5 mM beta-mercaptoethanol). The following four enzymatic reactions were then performed in 80 μL 1xPNK buffer (omitting the Nonidet P40 substitute), to ligate 3' and 5' adapters onto RNA fragments. After each reaction, beads were washed 1x in WBI and 3x in 1xPNK: (i) Alkaline phosphatase treatment (30 min, 37 °C): 8 U TSAP, 80 U RNasIN. (ii) 3' linker ligation (overnight, 16 °C): 0.1 nmol miRCat-33 DNA linker, 40 U T4 RNA Ligase 1, 80 U RNasIN, 12.5 % (v/v) PEG8000. (iii) 5' phosphorylation (1 hr, 37 °C): 40 U T4 PNK, 2 μL γ32P-ATP (after 30 min, add 1 μL 100 mM rATP and an additional 20 U T4 PNK). (iv) 5' linker ligation (overnight, 16 °C): 0.2 nmol 5' linker, 40 U T4 RNA Ligase 1, 1.25 mM rATP, 80 U RNasIN, 12.5 % (v/v) PEG8000. After the final reaction, beads were washed 3x in WBII (50 mM Tris-HCl pH 7.8, 50 mM NaCl, 0.1 % Nonidet P40 substitute, 5 mM beta-mercaptoethanol), resuspended in 30 µL 1x NuPAGE LDS sample buffer, heated at 95 °C for 2 min, and the eluate quickly removed and loaded onto a NuPAGE 4-12 % polyacrylamide gel. The gel was run at 100 V for ~1 hr, then protein:RNA complexes transferred to Hybond-C extra nitrocellulose membrane (Amersham) at 150 V for 1.5 hr using a wet transfer system and NuPAGE transfer buffer with 15 % methanol. The membrane was then briefly dried, exposed to BioMax MS film (4 hr to overnight) and the region corresponding to the protein:RNA complex cut out. The membrane slice was then incubated in 400 μL WBII with 1 % (w/v) SDS, 5 mM EDTA and 100 μg Proteinase K at 55 °C for 2 hr. The solution was then removed to another tube, 50 µl 3M NaAc pH 5.2 and 500 µl of 1:1 phenol:chloroform mix added, and the mixture vortexed then centrifuged at 14,000 xg for 20 min. The top phase was transferred into a new tube and 1 ml ethanol and 20 μg glycogen added. The solution was stored at -20 °C overnight to precipitate RNA, then centrifuged at 14,000 xg for 1 hr. The pellet was washed once with 70 % ethanol and allowed to briefly air dry, before resuspending in 11 μL water + 1 μL 10 μM miRCat-33 RT oligo + 1 μL 10 mM dNTP mix. The solution was heated to 80 °C for 3 min, snap cooled on ice for 5 min, then the following mix added: 4 μL 5x first strand buffer (SSIII kit) + 1 μL 100 mM DTT (SSIII kit) + 1 μL recombinant RNasIN. After incubating for 3 min at 50 °C, 200 U of SuperScript III was added and the reverse transcription allowed to proceed for 1 hr at 50 °C. The reaction was stopped by heating to 65 °C for 15 min, then RNA digested with 10 U RNase H at 37 °C for 30 min. PCR reactions (80 μL) were then prepared, each with 2 μL cDNA, 10 pmol P5, 10 pmol PE, 12.5 nmol each dNTP and 2.5 U LA Taq. Typically, we ran five PCR reactions and then concentrated the products by ethanol precipitation before resolving on a 3 % metaphor agarose gel in 0.5x TBE. A smear corresponding to the size of the two adapters plus inserts (total size ~120-300 bp) was then excised, and DNA extracted using the MinElute gel extraction kit, eluting in 20 μL water. If the experiment was successful, we repeated the PCRs with the remaining half of the cDNA. The CRAC protocol is referred to as the “long” protocol. For some samples, a shorter version of the protocol was used, which did not affect library content. The shorter version omits radiolabelling (using cold rATP instead), SDS-PAGE and transfer to nitrocellulose. Instead, after 3' linker ligation, beads were washed and added directly to 400 μL WBII with 1 % (w/v) SDS, 5 mM EDTA and 100 μg Proteinase K. This version of the CRAC protocol is referred to as the “short” protocol.