title: GSM1186350: SD-nitrogen polyA RNA, light fraction, gPAR-CLIP, Replicate 1; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: Crosslinked cells were resuspended in polysome lysis buffer (20 mM HEPES pH 7.5, 140 mM KCl, 1.5 mM MgCl2, 1% Triton X-100, 1X Complete Mini Protease Inhibitor EDTA-free (Roche 1 836 170), 0.2 U/µL SUPERase·In (Invitrogen AM2696)), mixed with ½ volume of acid-washed glass beads, and lysed by vortexing four times at 4°C, 1 min each with 1 min incubation on ice in between. Cell debris was removed by centrifugation for 5 min at 1,300Xg at 4°C. Supernatant was cleared by 20,000Xg spin for 10 min at 4°C. 15-50% (w/v) sucrose density gradients were prepared in Beckman polycarbonate centrifugation tubes (11 X 34 mm) by sequentially layering and freezing 0.24 mL of 50%, 41.25%, 32.5%, 23.75% and 15% sucrose dissolved in polysome gradient buffer (20 mM HEPES pH 7.5, 140 mM KCl, 5 mM MgCl2). Gradients were thawed overnight at 4°C before use. 100 µL of clarified lysate was loaded on top of a gradient, centrifuged for 1 hr at 54,000 rpm at 4°C using a TLS-55 rotor in an Optima MAX-E ultracentrifuge (Beckman Coulter). The top 600 µL of the gradient was recovered and supplemented with 2 µL of SUPERase·In (20 U/µL). 60 µL of freshly prepared 10mM EZ-Link NHS-SS-Biotin (Pierce 21441) dissolved in dimethylformamide was added to the recovered lysate and incubated on a Nutator for 2 hr at 4°C. 50 µL of 5 M NaCl was added to increase the total salt concentration to 0.5 M. Biotinylated lysate was mixed with 1 mg of oligo(dT)25 magnetic beads (NEB S1419S), then incubated on a Nutator for 30 min at 4°C. Beads were washed 4 times with ice-cold hybridization buffer (10 mM HEPES pH 7.5, 0.5 M NaCl, 1 mM EDTA). RNAs were eluted by incubating beads with 500 µL of elution buffer (10 mM HEPES pH 7.5, 1 mM EDTA) and heating at 65°C for 3 min. Eluted sample was transferred to a new tube and mixed with 55 µL of 10XPBS. PolyA-selected samples were mixed with 1 mg of streptavidin M280 Dynabeads (Invitrogen 112-05D) and incubated on a Nutator for 30 min at 4°C. Beads were washed 3 times with 1XPBS, then incubated with 20 µL of 50 U/µL RNase T1 (Fermentas EN0541, 1:20 dilution in 1XPBS) at 22°C for 15 min on an Eppendorf Thermomixer (15 sec shaking at 1,000 rpm followed by a 2 min rest interval), followed by 5 min incubation on ice. Beads were washed twice with wash buffer (1XPBS, 0.1% SDS, 0.5% deoxycholate, 0.5% NP-40), twice with high-salt wash buffer (5XPBS, 0.1% SDS, 0.5% deoxycholate, 0.5% NP-40), and twice with 1XPNK buffer (50 mM Tris pH 7.4,10 mM MgCl2, 0.5% NP-40). Beads were incubated with 20 µL of CIP mix (50 mM Tris pH 7.9, 100 mM NaCl, 10 mM MgCl2, 0.5 U/µL CIP (NEB M0290S)) at 37°C for 15 min, with 15 sec shaking at 1,000 rpm followed by a 2 min rest interval on a Thermomixer. After CIP treatment, beads were washed twice with 1XPNK+EGTA buffer (50 mM Tris pH 7.4, 20 mM EGTA, 0.5% NP-40) and twice with 1XPNK buffer. Beads were incubated with 20 µL of ligation mix (50 mM Tris pH 7.4, 10 mM MgCl2, 0.5 mM DTT, 2 µM Pre-adenylated 3’ DNA linker, 25% PEG-8000, 10 U/µL T4 RNA ligase 2, truncated K227Q (NEB M0351S)) at 16°C overnight (≥16 hr), with 15 sec shaking at 1,000 rpm followed by a 2 min interval on a Thermomixer. After linker ligation, beads were washed 3 times with 1XPNK+EGTA buffer. Beads were mixed with 12 µL of 1XPNK+EGTA buffer, 3 µL of freshly made 1M DTT and 15 µL of 4X NuPAGE LDS sample buffer (Invitrogen NP0007), and incubated at 70°C for 10 min. Beads were removed, and the supernatant was loaded onto NuPAGE 4-12% Bis-Tris gel (Invitrogen NP0335BOX) and run at 150 V for 35 min. Gel was transferred to Protran BA 85 nitrocellulose membrane (pore size 0.45 µm, Whatman 10402594) using Novex wet transfer at 30 V for 1 hr. A broad band from 31 kDa up to the top of the gel was excised, cut into small pieces, and transfered into a microfuge tube. Excised membranes were incubated with 500 µL of 4 mg/mL Proteinase K prepared in 1X PK buffer (100 mM Tris pH 7.5, 50 mM NaCl, 10 mM EDTA) for 20 min at 37°C on a Thermomixer. 500 µL of 7M urea prepared in 1X PK buffer were added to the tube followed by another 20 min incubation at 37°C. Proteinase K digestion reaction was mixed with 1 mL of Phenol:Chloroform:Isoamyl Alcohol 25:24:1 (Sigma-Aldrich P2069) by vortexing and spun for 5 min at 20,000Xg. Liquid phase was transferred to a new tube, mixed with 125 µL of 3 M NaOAc, 2.5 mL of 100% ethanol and 1 µL of 15 mg/mL glycoblue (Invitrogen AM9516), and precipitated for 2 hr at -80°C. RNAs were collected by centrifugation for 20 min at 20,000Xg at room temperature followed by 2 washes with cold 75% ethanol. RNA pellets were air-dried briefly, resuspended in 10 µL of PNK mix (70 mM Tris pH 7.6, 10 mM MgCl2, 5 mM DTT, 1 mM ATP, 1 U/µL T4 polynucleotide kinase (NEB M0201S), 1 U/µL SUPERase·In), and incubated at 37°C for 30 min. The reaction was combined with 90 µL of H2O and 100 µL of Phenol:Chloroform:Isoamyl Alcohol 25:24:1, mixed well, and spun for 5 min at 20,000Xg. The liquid phase was mixed with 12.5 µL of 3 M NaOAc, 250 µL of 100% ethanol, 1 µL of 15 mg/mL glycoblue and precipitated for 2 hr at -80°C. RNAs were collected by centrifugation for 20 min at 20,000Xg at room temperature, followed by 2 washes with cold 75% ethanol. RNA pellets were resuspended in 10 µL of ligation mix (50 mM Tris pH 7.5, 10 mM MgCl2, 10 mM DTT, 1mM ATP, 0.1 mg/mL BSA, 2 µM 5’ RNA linker, 1 U/µL T4 RNA ligase (Fermentas EL0021), 1 U/µL SUPERase•In, 10% DMSO) and incubated at 15°C for 2 hr. Ligation reaction was terminated by adding 10 µL of 2X formamide gel loading buffer (Invitrogen AM8546G), heated for 2 min at 70°C, and then quickly chilled on ice. Samples were loaded onto a 6% TBE UREA gel (Invitrogen EC6865BOX) and run at 150 V for 45 min. After staining with 1X Sybr Gold Stain (Invitrogen S-11494), a gel piece corresponding to 70-90 nt RNA (80-100 nt ssDNA) was excised, crushed, and soaked in 400 µL of 0.3 M NaOAc overnight at room temperature. After removing gel pieces, the solution was combined with 1 mL of 100% EtOH and 1 µL of 15 mg/mL glycoblue and precipitated for 2 hr at -80°C. RNAs were collected by centrifugation for 20 min at 20,000Xg at room temperature, followed by two washes with cold 75% ethanol. After brief drying, RNAs were resuspended in 15 µL of H2O. 10 μL of ligated RNA was combined with 2 μL of 5 µM RT primer, heated at 65°C for 5 min, and then quickly chilled on ice, followed by the addition of 1 µL of 10 mM dNTP, 1 μL of 0.1 M DTT, 4 μL of 5X First strand buffer, 1 μL of SUPERase•In (20 U/µL) and 1 µL of SuperScript III Reverse transcriptase (Invitrogen 18080-093, 200 U/µL). RT reaction was kept at 50°C for 45 min, 55°C for 15 min and 90°C for 5 min. A test PCR was performed with 2.5 µL of RT product in 50 µL PCR mix: 1X AccuPrime PCR buffer I, 0.5 µM P5 long primer, 0.5 µM P7 primer, 0.2 µL AccuPirme Taq High Fidelity (Invitrogen 12346-086, 5 U/µL). PCR was carried out with an initial 3 min denaturation at 98°C, followed by 14-22 cycles of 80 sec denaturation at 98°C, 90 sec annealing and extension at 65°C, and termination with a final 5 min extension at 65°C. 15 µL PCR product was collected after 14, 18, and 22 cycles and analyzed on a 10% TBE gel (Invitrogen EC6275BOX) at 150 V for 1 hr to determine the optimal amplification cycles (the lowest cycle number required to generate 96-116 bp amplicons detected by Sybr Gold staining). A 50 µL PCR reaction was carried out with the determined cycle number. Amplicons were purified using DNA clean and concentrator-5 (Zymo D4013), run on 10% TBE gels at 150 V for 1 hr and stained with Sybr Gold. A gel piece corresponding to 96-116 bp DNA was excised, crushed, and soaked overnight in 400 µL 0.3 M NaOAc at room temperature. After removing gel pieces, the solution was combined with 1 mL of 100% EtOH and 1 µL of 15 mg/mL glycoblue and precipitated for 2 hr at -80°C. DNAs were collected by centrifugation for 20 min at 20,000Xg at ro
attributes.tag: GEO Accession
attributes.value: GSM1186350