GSM1440303: LARP1 PAR-CLIP PP242; Homo sapiens; RIP-Seq
Crosslinked cell pellets were collected by scrapping and lysed with 600μl PAR-CLIP lysis buffer (0.1% SDS, 0.5% deoxycholate, 0.5% NP-40 in PBS without Mg2+, Ca2+) on ice for 10 minutes. To remove DNA, 10μl of RQ1 DNase was added to each tube. Tubes were incubated at 37°C for 10 min with gentle rocking. Afterwards, 1U/µl of RNase T1 was supplemented, and the lysates were incubated at room temperature for 15 min with gentle rocking. Lysates were spun at 4°C for 10 min at 14,000 rpm. The soluble fractions were incubated with LARP1 antibody-Dynabeads Protein A for 1 hr at 4°C. Immunoprecipitates were collected on the magnetic stand and were washed three times with IP-wash buffer (50mM HEPES-KOH [pH 7.5], 300mM KCl, 0.05% NP-40, 0.5mM DTT, and EDTA-free protease inhibitor). Immunoprecipitates were resuspended in 40μl of IP-wash buffer containing 50U/μl RNase T1 and incubated at room temperature with gentle rocking for 15 min followed by incubation on ice for 5 min. Immunoprecipitates were washed with high-salt wash buffer (50mM HEPES-KOH [pH 7.5], 500mM KCl, 0.05% NP-40, 0.5mM DTT, EDTA-free protease inhibitor), three times with the PAR-CLIP lysis buffer, twice with high-salt wash buffer (0.1% SDS, 0.5% deoxycholate, 0.5% NP-40 in 5x PBS without Mg2+, Ca2+), and twice with PNK (polynucleotide kinase) buffer (50mM Tris-Cl pH 7.4, 10mM MgCl2, 0.5% NP-40). For visualization of crosslinked RNAs, immunoprecipitates were incubated in 40μl of PNK mixture (1μl P32γATP, 4μl 10x PNK buffer [NEB], 2μl T4 PNK enzyme [NEB], 33μl water) for 30 min at 37°C. Labeled immunoprecipitates were washed three times with PNK buffer and resuspended with 30μl of 2x NUPAGE LDS sample buffer (15μl 1x PNK with 15μl Novex LDS sample buffer). Denatured samples were resolved in 4-12% NUPAGE Bis-Tris gel and transferred to nitrocellulose membrane at 30 V for 1 hr using NuPAGE transfer buffer. The membrane was expose to X-ray film at -80°C for 4 hr to visualize crosslinked RNAs. RNA pellets were air-dried briefly, resuspended in 10 µL of PNK mix (70mM Tris pH 7.6, 10mM MgCl2, 5mM DTT, 1mM ATP, 1U/µL T4 polynucleotide kinase, 1U/µL SUPERase·In), and incubated at 37°C for 30 min. The reaction was combined with 90µL H2O and 100µL Phenol:Chloroform:Isoamyl Alcohol 25:24:1, mixed well, and spun for 5 min at 20,000Xg. The liquid phase was mixed with 12.5µL of 3M NaOAc, 250µL of 100% ethanol, 1µL of 15mg/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 10µL of ligation mix (50mM Tris pH 7.5, 10mM MgCl2, 10mM DTT, 1mM ATP, 0.1mg/mL BSA, 2µM 5’RNA linker, 1U/µL T4 RNA ligase, 1U/µL SUPERase•In, 10% DMSO) and incubated at 15°C for 2 hr. Ligation reaction was terminated by adding 10µL of 2X formamide gel loading buffer, heated for 2 min at 70°C, and then quickly chilled on ice. Samples were loaded onto a 6% TBE UREA gel and run at 150 V for 45 min. After staining with 1X Sybr Gold Stain, a gel piece corresponding to 70-90nt RNA (80-100nt ssDNA) was excised, crushed, and soaked in 400µL of 0.3M NaOAc overnight at room temperature. After removing gel pieces, the solution was combined with 1mL of 100% EtOH and 1µL of 15mg/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 15µL of H2O. 10μL of ligated RNA was combined with 2μL of 5µM RT primer, heated at 65°C for 5 min, and then quickly chilled on ice, followed by the addition of 1µL 10mM dNTP, 1μL of 0.1 M DTT, 4μL of 5X First strand buffer, 1μL 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 AccuPrime Taq High Fidelity (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 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, 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 room temperature, followed by two washes with cold 75% ethanol. After brief drying, amplicons were resuspended in 20 μl of H2O. Purified amplicons (5 μl) were used to seed a second round of PCR in 50 μl: 1× Accu- Prime PCR buffer I, 0.5 μM Illumina Primer A, 0.5 μM Illumina Primer B, 0.2 μl AccuPirme Taq High Fidelity for 6 to 12 cycles. Second PCR amplicons were purified with DNA clean and concentrator-5.
GEO Accession
GSM1440303