Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: We re-suspended frozen pellets in 1 mL of RIPA lysis buffer (50mM Tris (pH 8), 150 mM KCl, 0.1% SDS, 1% Triton-X, 5 mM EDTA, 0.5% sodium deoxycholate,0.5 mM DTT (add fresh) + protease inhibitor cocktail (Thermo Scientific, PI-87785) + 100 U/ml RNaseOUTTM (Life Technologies , 10777-019)). We incubated cells at 4°C for 10 minutes before lysing on a Branson® digital sonifier using 10% amplitude for 0.7 seconds on and 1.3 seconds off at 30 second intervals for a total of 90 seconds. We used chilled tube holders and swapped them out between shearing runs to reduce temperature elevation. After lysis, we spun the lysate at 4°C max speed for 10 minutes. We collected supernatant and diluted by adding equal volume of fRIP binding/wash buffer (150 mM KCl, 25 mM Tris (pH 7.5), 5 mM EDTA, 0.5% NP-40, 0.5 mM DTT (add fresh), 1X PIC (add fresh), 100 U/mL RNaseOUT (add fresh)). At this point, we removed 50 μl of lysate for input sample and stored at -20°C for later RNA purification and library construction. After dilution, we clarified the lysate by passage through a 0.45 μM syringe filter. We then “pre-cleared” filtered lysate by incubating with Dynabeads® Protein G (Life Technologies cat#10004D) at a concentration of 25 μl of beads per 5 million cells for 30 minutes at 4°C with slow rotation. We flash froze pre-cleared lysate in 1 mL aliquots of ~5 million cells and stored it at -80 °C. For fRIP, we thawed lysate on ice and added 6 μg of HuR antibody (Santa Cruz, sc-5483). After addition of antibody, we rotated lysate at 4°C for 2 hours before adding 50 μl of Dynabeads® Protein G. We rotated beads and lysate at 4°C for 1 hour before washing 2X with 1 mL of fRIP binding/washing buffer + 1X PIC and 100 U/mL RNaseOUT. After the final wash, we removed the supernatant and froze and stored the beads at -20°C. We re-suspended the frozen beads in 56 μl of RNase-free water and added 33uL of 3X reverse-crosslinking buffer (3X PBS (without Mg or Ca), 6% N-Lauroyl Sarcosine, 30 mM EDTA, 15 mM DTT (add fresh)), 10 μl of Proteinase K (Life Technologies, cat #AM9516), and 1 μl of RNaseOUT to both the re-suspended beads and input sample. We performed protein degradation and reverse-crosslinking for 1 hour at 42°C, then another 1 hour at 55°C. We added beads and reaction buffer to 1 mL of TriZol (Life Technologies, 15596-026). After agitation, we added 200 μl of chloroform followed by ~15 seconds of vigorous agitation and a 20 minute microcentrifuge spin at 4°C max speed. We collected the aqueous layer, added it to 750 μl of ethanol + 1 μl GlycoBlue™, and ran it over a Qiagen RNeasy® min-elute column (Qiagen, cat #74204). We extracted RNA using the buffer RWT/3X isopropanol modification detailed in “Appendix B: Optional On-Column DNase Digestion…” of the Qiagen miRNeasy® Mini Handbook. We eluted RNA in 15μl of RNase-free water. to remove ribosomal RNA, we fed ≥ 70 ng of input and fRIP RNA into the Ribo-Zero™ Magnetic Gold Kit (Epicentre, cat #MRZG12324) followed by a cleanup using Agencourt RNAClean XP beads (Beckman Coulter, cat #A63987) and elution with 19.5 uL of Elute, Prime, Fragment mix from the TruSeq RNA Sample Preparation Kit (Illumina, cat #RS-122-2001). We performed library construction per the vendor’s instructions, starting with the “Incubate RFP” step. We pooled the resulting cDNA libraries and subjected them to paired-end sequencing on an Illumina HiSeq 2500 at a depth of 31 base pairs per read.