GSM2454303: fRIP_WT-input rep 2; Mus musculus; RIP-Seq
P12 testes were cross-linked in 0.1% formaldehyde at room temperature for 10 minutes. Crosslinking reaction was quenched by adding glycine to a final concentration of 125mM and incubating for 5 minutes at room temperature. Cross-linked testes were washed 2x in 4C PBS, flash frozen and stored at -80ºC. Frozen testes were resuspended in RIPA lysis buffer (50mM Tris-HCl pH8.0, 150mM KCl, 0.1% SDS, 1% Triton-X-100, 5mM EDTA, 0.5% sodium deoxycholate, 0.5mM DTT, 1x Complete protease inhibitor, and 100U/mL RNasOUT). Testes were mechanically disrupted in lysis buffer and incubated for 15 minutes at 4C then sonicated using a Bioruptor (1 cycle, 30 sec on/off, medium setting at 4C). After lysis, lysates were centrifuged for 10 minutes at max speed at 4C. The supernatant was collected and diluted with an equal volume of binding/wash buffer (25mM Tris-HCl pH7.5, 150mM KCl, 5mM EDTA, 0.5% NP-40, 0.5mM DTT, 1x Complete protease inhibitor, and 100U/mL RNasOUT). Lysates were precleared by incubation with uncoupled Protein A Dynabeads (Invitrogen) for 1 hour at 4C. Following preclearing, 10% of the lysate was saved for input and stored at 4C, and the remaining lysate was added to Protein A Dynabeads chemically crosslinked to rabbit anti-YTHDC2 (Bethyl laboratories) with dimethylprimelimidate (DMP, Sigma) (5.4 mg DMP per mL 0.2M triethanolamine) per manufacture’s instructions, and incubated for 4 hours at 4C. After incubation, samples were washed 4x with binding/wash buffer for 10 minutes at 4C. Beads and Input samples for 2 biological replicates of both wild type and Ythdc2-/- were suspended in PK buffer (10mM Tris pH 7.0, 100mM NaCl, 1mM EDTA, 0.5% SDS), 5uL Proteinase K and 1uL RNasOUT (100uL total volume). Samples were incubated for 1 hour at 42C and then 1 hour at 55C. RNA was isolated using TRIzol (Life Technologies), according to manufacturer’s instructions. Ribosomal RNA was removed from the Input and fRIP samples using the RiboMinus Eukaryote System v2 Kit (Ambion) following manufacture’s instructions. Library preparation for high-throughput sequencing was performed using a TruSeq RNA Sample preparation Kit (Illumina) per manufacture’s instruction, except RNA was not fragmented with FPF solution.