GSM2342257: HuR.rep2 DO-RIP; Homo sapiens; OTHER
HEK293 cells were lysed on ice by resuspending and repeated pipetting with 1.5 cell pellet volumes of polysome lysis buffer (10 mM HEPES pH 7.0, 100 mM KCl, 5 mM MgCl2, 5 mM CaCl2, 1X cOmpleteTM protease inhibitor cocktail (Roche), 100 Units/ml RNaseOUT (Life Technologies), 0.5% IGEPAL, and 1 mM DTT). Lysates were incubated on ice for 5 min and then stored at -80 °C. These lysates were thawed on ice and then centrifuged at 4 °C to clear the cell debris. The cleared lysates were digested in 300 μl volumes (the volume used in the optimization experiment) using 30 gel Units MNase/μg RNA. As described above, digestions were performed by incubating the lysate with micrococcal nuclease for 5 min at 30 °C, and inactivating the enzyme by adding EGTA to a final concentration of 10 mM. Digested lysates were pooled and then evenly added to immunoprecipitation reactions with either magnetic protein G beads coupled to mouse anti-HuR (3A2) antibodies, or to normal mouse serum antibodies. Immunoprecipitations were performed at 4 °C for 2 h with constant mixing, and then the bead-antibody complexes washed 4 times with ice-cold NT2 buffer. RNA fragments from the IP were extracted, dephosphorylated, and radiolabeled as described above. Since these RNA fragments were going to be used for cDNA libraries, 10 nmol of non-radioactive ATP was added to the radiolabeling reaction after the 30 min incubation with [γ-P32]ATP, and incubated for an additional 10 min at 37 °C to ensure complete labeling of the ends. As described above, end-labeled fragments were separated by polyacrylamide gel electrophoresis, and the gel visualized using a phosphor-imaging scanner. RNA fragments corresponding to 25-70 nucleotides in length were cut from the gel, and extracted overnight by constantly mixing the gel slices in 400 μl of 0.4 M NaCl at 4 °C. The supernatant containing the radiolabeled RNA fragments was transferred to a new microcentrifuge tube. 200 μl of 0.4 M NaCl was then added to the gel slices and constantly mixed at 4C for 1h to wash. The supernatant from the wash was pooled with the supernatant from the overnight extraction, and supernatants were precipitated with 1ml 100% EtOH and glycogen at -20 °C for at least 1h or -80 °C for 30 min. The RNA fragments were centrifuged at 17,000 g, 4 °C for 20 min, washed once with 80% ethanol, centrifuged at 10,000 g, 4 °C for 5 min, and air-dried. NEBNext small RNA library prep protocol (New England BioLabs); Adapters and Primers for HuR.1 and Neg.1 DO-RIP-seq libraries = 5' RNA adapter: GUUCAGAGUUCUACAGUCCGACGAUC; 3' DNA adapter: /5rApp/NNNNNTCGTATGCCGTCTTCTGCTTG/3ddC/; 5' PCR DNA primer: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA; 3' PCR DNA primer: CAAGCAGAAGACGGCATACGA. Adapters and Primers for HuR.2 and Neg.2 DO-RIP-seq libraries = 5' RNA adapter: GUUCAGAGUUCUACAGUCCGACGAUC; 3' DNA adapter: /5rApp/NNNNNTCGTATGCCGTCTTCTGCTTG/3ddC/; 5' PCR DNA primer: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA; 3' PCR DNA primer for HuR.2: GTCAAGCAGAAGACGGCATACGA; 3' PCR DNA primer for Neg.2: ACCAAGCAGAAGACGGCATACGA. Adapters and Primers for HuR.3 and Neg.3 DO-RIP-seq libraries = 5' RNA adapter: GUUCAGAGUUCUACAGUCCGACGAUC; 3' DNA adapter: /5rApp/NNNNNAGATCGGAAGAGCACACGT/3ddC/; 5' PCR DNA primer: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA; First strand synthesis DNA primer: ACGTGTGCTCTTCCGATCT; 3' HuR.3 Index primer: CAAGCAGAAGACGGCATACGAGATGTGACGAGTTCATTGGCACGTGTGCTCTTCCGATCT; 3' Neg.3 Index primer: CAAGCAGAAGACGGCATACGAGATGTGACGAGTTCTACAAGACGTGTGCTCTTCCGATCT
GEO Accession
GSM2342257