title: GSM1612328: Pfk2p PAR-CLIP-seq; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Cells were lysed in 1XPBS, 0.5% NP-40, 1X Complete Mini Protease Inhibitor, EDTA-free and cleared by sequential spins at 1,300Xg for 5 min and 20,000Xg for 10 min at 4°C. Clarified lysate was passed through a Costar Spin-X filter, mixed with RNase T1 to 1 U/µL, and incubated at 22°C for 15 min followed by 5 min incubation on ice. Lysate was then directly mixed with IgG magnetic beads (prepared by coupling rabbit IgG to Dynabeads M-270 Expoxy) to pull down Pfk2p::TAP. RNase T1 digestion, CIP treatment, and 3’ DNA linker ligation were performed as follows: Beads were washed 3 times with 1XPBS, then incubated with 20 µL of 50 U/µL RNase T1 (1:20 dilution in 1XPBS) at 22°C for 15 min on Eppendorf Thermomixer (15 sec shaking at 1,000 rpm followed by 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) at 37°C for 15 min, with 15 sec shaking at 1,000 rpm followed by 2 min rest interval on 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) at 16°C overnight (≥16 hr), with 15 sec shaking at 1,000 rpm followed by 2 min interval on Thermomixer. 5’ end phosphorylation was performed on-bead in 20 µL of PNK mix (70 mM Tris pH 7.6, 10 mM MgCl2, 5 mM DT, 1 µL P32 rATP (6000 Ci/mmol 10 mCi/mL), 1 U/µL T4 polynucleotide kinase) and incubated at 37°C for 15 min. 2 µL of 10 mM ATP was added to the mix, and the reaction was incubated for 10 min. Beads were removed, and the supernatant was loaded onto NuPAGE 4-12% Bis-Tris gel and run at 150 V for 35 min. Gel was transferred to Protran BA 85 nitrocellulose membrane (pore size 0.45 µm) using Novex wet transfer at 30 V for 1 hr. Crosslinked RNAs were visualized by autoradiography, and the corresponding Pfk2p band was excised and 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 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 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, 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, 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 , heated for 2 min at 70°C, and then quickly chilled on ice. Samples were loaded onto 6% TBE UREA gel and run at 150 V for 45 min. After staining with 1X Sybr Gold Stain, 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 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, 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, 5 U/µL. PCR was carried out with 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 at 150 V for 1 hr to determine optimal amplification cycles (the lowest cycle number required to generate 96-116 bp amplicons detected by Sybr Gold staining). 50 µL PCR reaction was carried out with the determined cycle number. Amplicons were purified using DNA clean and concentrator-5, run on 10% TBE gels at 150 V for 1 hr and stained with Sybr Gold. 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, 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 room temperature, followed by 2 washes with cold 75% ethanol. After brief drying, amplicons were resuspended in 20 µL of H2O. 5 µL of purified amplicons were used to seed a second round of PCR in 50 µL: 1X AccuPrime PCR buffer I, 0.5 µM Illumina Primer A, 0.5 µM Illumina Primer B, 0.2 µL AccuPirme Taq High Fidelity for 6-12 cycles. Second PCR amplicons were purified with DNA clean and concentrator-5 and sequenced on an Illumina HiSeq 2000 sequencer.
attributes.tag: GEO Accession
attributes.value: GSM1612328