GSM4654284: Quaking KO, time 10, replicate 1; Mus musculus; RIP-Seq
The iCLIP2 protocol was carried out according to Buchbender et al., 2019. Briefly, cells were washed with 5 ml PBS and 6 ml of ice-cold PBS were added to cells growing in a 10cm plate. The plate was then placed on ice. The lid was removed and the cells were irradiated once with 150 mJ/cm2 in a UV-C crosslinker (CL 1000 Ultraviolet Crosslinker, UVP) at 254 nm. Cells were harvested by scraping and cell suspension was transferred in a microtube, spinned at 1,500 xg (4,000 rpm) for 1 min at 4°C. Snap frozen pellets were stored at -80°C until use. To prepare the immunoprecipitation, a volume of 100 µL of protein G Dynabeads per experiment was added to a microtube. Beads were washed twice with lysis buffer and resuspended in 100 µl of lysis buffer with 2 to 10 µg antibody per experiment. Bead-antibody conjugation occurred by rotating tubes at room temperature for 30-60 min (until lysate was ready). The beads were washed once with high-salt wash buffer, twice with lysis buffer and resuspended in 100 µL lysis buffer with added protease inhibitor. In parallel, cell pellets were resuspended in 1 ml lysis buffer supplemented with protease inhibitors An optimized RNase I dilution in lysis buffer was prepared and added to the lysate together with 2 µl Turbo DNase. RNA was digested for 3 min shaking at +37°C and 1,100 rpm. After incubation the reaction was transferred to ice for 3 minutes and centrifuged at +4°C at full speed 21,100 xg (14,800 rpm) for 10 min and the supernatant transferred to a new 1.5 ml tube. A volume of 500 µl of lysate was loaded onto a Proteus mini clarification spin column and centrifuged at +4°C at 16,000 xg (12,900 rpm) for 1 min. The flow-through was moved to a new tube. The step was repeated with second half of the lysate. To carry out the immunoprecipitation, the beads were added to cell extracts and the sample were incubated on a rotating wheel for 2 hours at +4°C. Supernatant was discarded and the beads were washed twice with high-salt wash buffer and twice with PNK buffer in order to be resuspended in 1 ml PNK buffer. To promote RNA 3' end dephosphorylation, the supernatant was carefully removed and the beads were resuspended in 20 µl of the following mix:8 µl water, 5 µl 4X ligation buffer 0.5 µl  RNase inhibitor, 1.5µl Pre-adenylated L3-App (20 µM) 4µl PEG400, 1µl  T4 RNA ligase. The samples were incubated overnight at +16°C in a thermomixer at 1,100 rpm and a volume of 500µl PNK buffer was added. Samples were washed twice with 1 ml high-salt wash buffer, twice with 1ml PNK buffer and left in 1 ml of the second wash. The samples were the loaded on a 4-12% NuPAGE Bis-Tris gel according to the manufacturer's instructions (Life Technologies) immerged in 1X MOPS running buffer. The run vas performed for 50 min at 180 V. Protein-RNA complexes were dry transferred from the gel to a nitrocellulose membrane and excised from the nitrocellulose membrane and the selected regions were cut in several pieces and placed into 1.5 ml tubes. A volume of 10µl proteinase K in 200µl PK buffer was added to the nitrocellulose pieces and the tubes were incubated in a thermomixer for 20 min at 37°C and 1,100rpm. Another 200µl of PK + urea buffer were added and the samples were incubate for further 20 min at +37°C and 1,100 rpm. The solution was collected and added together with 400µl phenol/chloroform to a 2 ml Phase Lock Gel Heavy tube. The tubes were then incubated for 5 min at +30°C shaking at 1,100rpm (do not vortex). In order to separate the phases the samples were spin for 5 min at 16,000 xg (12,900 rpm) at room temperature. The aqueous layer was transferred into a new tube and spin again for 1 min in order to be transferred again into a new tube. Precipitation was performed by adding 0.75µl GlycoBlue and 40µl 3M sodium acetate pH 5.5. After mixing 1ml 100% ethanol was added and the tubes were mixed by inverting several times and placed over night at -20°C. The tubes were centrifuged for 20 min at 21,100 xg (14,800 rpm) at +4°C in order to remove the supernatant and the pellet was washed with 0.9 ml of 80% ethanol, and centrifuged again for 5 min. After air-drying it for 3 min, the pellet was resuspended in 5µl water and transfer to a PCR tube in order to be retrotrascribed. Reverse transcription was promoted by adding the following reagents: 1µl primer RToligo (0.5 pmol/µl) and 1 µl dNTP mix 10 mM setting the temperature at +70°C for 5 min and +25°C hold until the RT mix (7µl water, 4µl of 5X RT buffer, 1 µl 0.1 M DTT, 0.5 µl RNase inhibitor, 0.5µl SuperScript III) was added. The following programme was used: +25°C for 5 min, +42°C  20 min, +50°C 40 min, +80°C 5 min, +4°C  hold. A volume of 1.65 µl 1 M NaOH was added and samples were incubated at 98°C for 20 min. Then 20 µl 1 M HEPES-NaOH pH 7.3 was added.  To clean up the samples from all the reagents, we used the MyONE clean-up. Briefly, a volume of 10μl MyONE Silane beads was considered per sample. Beads were magnetically attracted and washed with 500 μl RLT buffer and resuspended in 125 μl RLT buffer. A volume of 125 μl washed beads was added to each sample and mixed with an additional 150 μl of 100% ethanol.  After 5 min at room temperature, the sample were mixed once more by pipetting and incubated a second time for 5 min. Beads were magnetically attracted and the supernatant was discarded. Beads were resuspended in 1 ml 80% ethanol and transferred to a new tube. Again, beads were washed twice with 80% ethanol and incubated for 30 seconds at room temperature. The mix was briefly spin in a microcentrifuge and the supernatant was discarded. Beads were air-dried for 5 min at room temperature, resuspended in 5 μl water and incubate for 5 min at room temperature. The second adapter was added together with DMSO to the cDNA-bead solution and the mix was heated for 2 min at 75°C and immediately kept it on ice for >1 min:2 μl L##clip2.0 (10 µM), 1 μl 100% DMSO. The following ligation master-mix was prepared on ice: 0.3 µl water, 2.0 μl 10X NEB RNA ligase buffer (with DTT) , 0.2μl ATP, 100 mM, 9.0μl, 50% PEG 8000, 0.5 μl High conc. RNA Ligase. To ensure homogeneity, the ligation master-mix was mixed by vigorous stirring, pipetting and flicking. The mix was briefly centrifuged in a microcentrifuge. A volume of 12 µl of ligation master-mix was added to 8 µl sample-linker mix and mixed it thoroughly.  1 μl of RNA ligase was added to each sample (final volume: 21 µl), followed by stirring and the sample was incubated overnight at room temperature, shaking at 1,100 rpm.  Fresh MyONE Silane beads were added to the cDNA-bead mix. Therefore, 5μl of fresh MyONE Silane beads were magnetically attracted, discarding the supernatant and washed with 500μl of RLT buffer. Magnetic attraction was repeated and in order to resuspend the beads in 60µl RLT per sample. A volume of 60μl beads was added to the cDNA-bead slurry and mixed. Then 60μl of 100% ethanol were added and the solution was mixed by pipetting carefully. After 5 min at room temperature, the sample was mixed once more by pipetting and the 5 min incubation step was repeated a second time.   The beads were magnetically attracted and the supernatant was discarded. The beads were resuspended in 1 ml 80% ethanol and the mix was transferred to a new tube. This step was repeated other two times incubating the mix 30 seconds at room temperature in 80% ethanol. The mix was spin in a microcentrifuge and after the beads were magnetically attracted, the supernatant was discarded. After air-drying the beads for 5 min at room temperature, they were resuspended in 23μl of water and the mix was incubated for 5 min at room temperature. The beads were magnetically attracted and the eluate was added to the PCR mix of the next step. c-DNA was pre-amplified by adding the following PCR mix: 22.5 µl cDNA , 2.5 µl Primer mix of P5Solexa_s and P3Solexa_s, 10 µM each, 25.0 µl, 2X Phusion HF PCR Mast