GSM2839005: NES_Input_rep3; Homo sapiens; RIP-Seq
All buffers used during RNA isolation were supplemented to 0.1 U/uL RNaseOUT (Thermo Fisher), 1xEDTA-free proteinase inhibitor cocktail (Thermo Fisher) and 0.5 mM DTT, final. Labeled, crosslinked cell pellets were lysed by incubation in 1 mL ice-cold RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 5 mM EDTA), supplemented with 10 mM ascorbate and 5 mM Trolox, for 5 min at 4 ¡C with end-over-end agitation. Samples were then sheared using a Branson Digital Sonifier 250 at 10% amplitude for three 30-s intervals (0.7 s on + 1.3 s off), with 30-s resting steps between intervals. Samples were held in ice-cold metal thermal blocks throughout sonication. Lysates were then clarified by centrifugation at 15,000xg for 5 min at 4 ¡C, moved to fresh tubes and each diluted with 1 mL Native lysis buffer (NLB: 25mM Tris pH 7.4, 150 mM KCl, 0.5% NP-40, 5 mM EDTA), supplemented with ascorbate and trolox. ÒInputÓ samples corresponded to 20% of these lysates. To the remainder was added 50 uL of streptavidin-coated magnetic bead slurry (Pierce) that had been equilibrated by two washes in 1:1 RIPA:NLB. Samples were incubated for 2 h at 4 ¡C with end-over-end agitation. Beads were subsequently washed with the following series of buffers (1 mL each, 5 min per wash, 4 ¡C, with gentle end-over-end agitation): (1) RIPA buffer, supplemented with trolox and ascorbate, (2) RIPA buffer without radical quenchers, (3) high salt buffer (1 M KCl, 50 mM Tris, pH 8.0, 5 mM EDTA), (4) urea buffer (2 M Urea, 50 mM Tris, pH 8.0, 5 mM EDTA), (5) RIPA Buffer, (6) 1:1 RIPA: NLB, (7) NLB, and (8) TE (10 mM Tris, pH 7.4, 1 mM EDTA). Input and enriched RNAs were harvested and reverse-crosslinked by proteolysis in 100 uL of Elution Buffer (2% N-lauryl sarcoside, 10mM EDTA, 5mM DTT, in 1X PBS, supplemented with 200 ug proteinase K (Ambion) and 4 U RNaseOUT) at 42 ¡C for 1h, followed by 55 ¡C for 1h, as previously described. Eluted samples were cleaned up using Agencourt RNAClean XP magnetic beads (Beckman Coulter), following the manufacturer's 1.5 mL tube format protocol, and eluted into 85 uL H2O. Thereafter, contaminating DNA was removed by digestion with 5 U RQ1 RNase-free DNase I (Promega) in 100 uL of the manufacturer's supplied buffer (1X final concentration) at 37 ¡C for 30 min. Purified RNAs were again cleaned up using Agencourt RNAClean XP magnetic beads, as above, and eluted into 30 uL H2O. Purified RNA samples were depleted of ribosomal RNA using the Ribo-Zero Gold rRNA removal kit (Illumina), generally in accordance with the manufacturer's protocol. Briefly, RNA concentration and integrity were quantified on an Agilent 2100 Bioanalyzer, using ÒRNA PicoÓ and, where appropriate, ÒRNA NanoÓ kits. Samples were not concentrated prior to rRNA depletion, which can accommodate a maximum input volume of 17 uL. Therefore, samples with total input masses of 0-20 ng or 20-100 ng were mixed with 1 uL or 2 uL of Ribo-Zero rRNA Removal Solution, respectively, in 1x RiboZero Reaction Buffer, at a final volume of 20 uL. Reaction mixes were incubated at 68¡C for 10 minutes, followed by 25¡C for 5 minutes more, and thereafter added to 32.5 uL magnetic beads (90 uL bead slurry; washed with water and equilibrated in Magnetic Bead Resuspension Buffer, supplemented with RiboGuard RNase Inhibitor) by extensive pipetting. Binding reactions were incubated at room temperature for 5 minutes, gently vortexed for 5 seconds, and incubated for 5 minutes at 50¡C, in a thermocycler. The supernatant, containing rRNA-depleted RNA, was diluted in water to 50 uL final volume, cleaned up with 50 uL Agencourt RNAClean XP beads and eluted with 19.5 uL of Elute, Prime, Fragment mix from the TruSeq RNA sample preparation kit, v2 (Illumina). Thereafter, libraries were prepared using the TruSeq RNA sample preparation kit, according to the manufacturer's instructions, starting from ÒIncubate RFPÓ step.
GEO Accession
GSM2839005