GSM8781742: IGF2BP3 PAR-CLIP, Control for Arsenite samples, 2; Homo sapiens; OTHER
Five 15 cm (diameter) dishes of 60 % confluent cells per condition were put on ice, washed with ice-cold PBS (Sigma) and crosslinked with the UV light at 365nm with 0.15 J/cm2 and pelleted. Cell pellets were lysed in 1250 μL of ice-cold lysis buffer (25 mM HEPES pH 7.3, 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA, 10% Glycerol, 0.1% SDS, 0.2% Sodium Deoxycholate, 1x protease inhibitors cocktail, 0.1mM DTT), by incubation on ice for 15 min with intermittent vortexing and passing three times through the 27G needle. The lysate was clarified by centrifugation for 20 min at 20,000g at +4ºC. 1100 μL of the supernatant was taken for the in-lysate RNase digestion on a rotator for 15 min at room temperature. The RNase I (Ambion) was used at 0.1 or 0.05 U/μL and RNase T1 (Thermo Scientific) at 1 or 0.25 U/μL. For IP 50 μg of Proteintech anti-IGF2BP3 antibody (14642-1-AP, lot 00088732) or Proteintech IgG control (30000-0-AP) were coupled to the 200 μL of protein G Dynabeads (Invitrogen) in 1 mL of lysis buffer for 20 min rotating at room temperature, washed three times with 1 mL of the lysis buffer, resuspended in the original bead volume (200 μL) and added to 1 mL of the RNase treated lysate. The IP was incubated on a rotator at +4ºC for 4 hours, washed twice and resuspended in 1 mL of the lysis buffer. For the on-beads RNase treatment RNase I (Ambion) was added at 0.05 or 0.025 U/μL and RNase T1 (Thermo Scientific) at 1 or 0.25 U/μL. For RNase treatment, the samples were incubated on the rotator for 15 min at room temperature and cooled on ice for 5 min. Then the beads were washed three times with 1 mL of ice-cold high salt wash buffer (25 mM HEPES pH 7.3, 400 mM NaCl, 0.5% NP-40, 0.5 mM EDTA, 10% Glycerol, 1x protease inhibitors cocktail, 0.1mM DTT), with 3 min incubations on ice after each wash step. Library strategy: PAR-CLIP For dephosphorylation of the crosslinked RNA fragments, the beads were washed once with 1 mL of PNK wash buffer (20 mM Tris-HCl pH 6.5, 10 mM MgCl2, 0.2% Tween20, 0.1mM DTT), resuspended in 200 μL of dephosphorylation mix (1x PNK buffer pH 6.5 (70 mM Tris-HCl pH 6.5, 10 mM MgCl2, 1 mM DTT), 100 U T4 polynucleotide kinase (NEB), 20 U SUPERase·In (Invitrogen)) and incubated at 37 °C for 30 min with shaking at 1100 rpm. Afterwards, the beads were kept on ice and washed once with 1 mL of PNK wash buffer, incubated in 1 mL of high salt wash buffer for 5 min on ice and washed once more with 1 mL of PNK wash buffer. For 3' adapter ligation (ordered as /5Phos/NNNNNATCGTAGATCGGAAGAGCACACGTCTGAA/3AzideN/, pre-adenylated using 5' DNA Adenylation Kit (NEB) and conjugated to the IRdye-800CW-DBCO (LiCor) was via “click” chemistry at 37 °C for 2 hours as described in (Zarnegar et al. 2016), the beads were resuspended in 200 μL of the ligation reaction mix (20% PEG8000, 1x T4 RNA Ligase Reaction Buffer (NEB), 1000 U T4 RNA Ligase 2 truncated KQ (NEB), 50 pmol 5' pre-adenylated IR-dye conjugated DNA adapter and 20 U SUPERase·In) and incubated at 25°C for 3 hours with shaking at 1100rpm in the dark. Next, the beads were transferred on ice and washed once with 1 mL of PNK wash buffer, incubated in 1 mL of high salt wash buffer for 5 min on ice, resuspended in 1 mL of PNK wash buffer and transferred to the new low-bind RNase-free 1.5 mL tube. The PNK wash buffer was removed and the crosslinked protein-RNA complexes were eluted in 40 μL of 1x SDS sample buffer without DTT at +70ºC for 10 min. DTT at 20mM concentration was added to the collected eluates and the samples were heated at +70ºC for 10 min and stored at -80ºC. Next, the crosslinked protein-RNA complexes were resolved on a 10 well 4-12% NuPAGE Novex Bis-Tris gel (Invitrogen) in 1x MOPS-SDS running buffer (Invitrogen) at 200 V for 65 min. The gel was visualized at 800nm using LI-COR Odyssey CLx near infrared imager and RBP-RNA complexes were size selected using imager's grid. The bands corresponding to IGF2BP3-RNA complexes were excised from the gel and transferred to a low-bind RNase-free tube. For the samples with the RNase treatment conditions in-lysate and on-beads: RNase I 0.05 U/µl, RNase T1 1 U/µl proteinase K digestion was performed as described in (Acosta-Alvear et al. 2018). The gel piece was transferred to nuclease free water preequilibrated Pur-A-Lyzer Midi Dialysis Tube (MWCO 3.5 kDa, Sigma) with 200 μL 1x MOPS-SDS running buffer (Invitrogen) and electroelution was performed at 100 V for 2 h. The polarity of the electric current was reversed for 120 seconds, sample was transferred to the new 2 mL low-bind RNase-free tube with an equal volume of 2x Proteinase K buffer (100 mM HEPES pH 7.3, 100 mM NaCl, 20 mM EDTA, 2% SDS) and proteinase K (Invitrogen) at the final concentration of 1.2 mg/ml and incubated for 30 min at +55 ºC. For the samples with RNase treatment conditions: RNase I 0.05 U/µl in-lysate and 0.025 U/µl on-beads, RNase I 0.01 U/µl in-lysate and 0.025 U/µl on-beads, and RNase T1 0.25 U/µl in-lysate and on-beads gel extraction and protein digestion were performed as described in (Anastasakis et al. 2021). Gel pieces were transferred to 1.5 mL low-bind RNase-free tube, frozen at -80ºC, thawed at +55ºC and crashed with a single use RNase-free pellet pestle. Gel slurry was boiled in 2x proteinase K buffer with 50 mM DTT at +95ºC for 2 min and was subsequently incubated three times with fresh addition of proteinase K at 2.4 mg/ml, 1.5 mg/ml, and 2.4 mg/ml in 2x proteinase K buffer at +50 ºC for 30 min with shaking at 1100rpm. Extracted 3' adapter-ligated RNA fragments were separated from the gel by centrifugation at 10,000g for 10 min through Spin-X centrifuge filter (Costar). For all samples, RNA was extracted with 1 mL of acidic phenol – chloroform – IAA (25:24:1, pH 4.0), isopropanol precipitated, resuspended 1x TBE-Urea dye (Invitrogen), denatured by heating at +75ºC for 2 min and resolved on a 10 well 15% TBE-Urea gel (Invitrogen) in 1x TBE running buffer (Invitrogen) at 180 V for 70 min. The gel was visualized at 800 nm using LI-COR Odyssey CLx near infrared imager and the 3' adapter-ligated RNA fragments were size selected using imager's grid. Gel pieces were transferred to 1.5 mL low-bind RNase-free tube, frozen at -80º C, thawed at +55º C and crashed with a single use RNase-free pellet pestle. 3' adapter-ligated RNA fragments were extracted from the gel during overnight incubation rotating in 300 μL of RNA elution buffer at +4ºC (10mM Tris-HCl pH 7.0, 0.3 M NaOAc pH 5.5, 2mM EDTA, 20 U SUPERase·In). Extracted complexes were separated from the gel fragments by centrifugation at 10,000 g for 10 min through Spin-X centrifuge filter, ethanol precipitated and resuspended in 11.5 μL of nuclease-free water. For reverse transcription, 1 μL of 1 μM reverse transcription primer (/5Phos/RNAGATCGGAAGAGCGTCGTGTAGGGAAAGAG/iSp18/GTGACTGGAGTTCAGACGTGTGCTC) was added to the sample. The sample was incubated at +70 ºC for 10 min and transferred on ice. Then the components of the reverse transcription mix were added to the sample making final volume of 20 μL (1x Superscript IV buffer, dNTPs 0.2 mM each, 5 mM DTT, 10 U SUPERase·In, 200 U Superscript IV (Invitrogen)), the reaction was performed at +55 ºC for 20 min and inactivated at +80 ºC for 5 min. 2.2 µl of 1 M NaOH was added and RNA was hydrolyzed in 30 min incubation at 98 °C. The reaction was neutralized by addition of 2.2 µl of 1 M HCl. The reverse transcription reaction was purified on Oligo Clean & Concentrator columns (Zymo) according to the manufacturer's instructions and eluted in 6 µl of nuclease-free water. cDNA was mixed with 6 µl of 2x TBE-Urea dye), denatured by heating at +75ºC for 2 min and resolved on a 10 well 10% TBE-Urea gel (Invitrogen) in 1x TBE running buffer at 180 V for 70 min. The gel was stained with SYBR gold (Invitrogen), visualized and the reverse transcription product was separated from the unreacted primer and no-insert products. Gel extraction was performed similarly as for the 3' adapter-ligated RNA fragments using DNA elution buffer (10 mM Tris-HCl pH 8.0, 0.3 M NaOAc pH 5.5). After ethanol precipitation, the pellet was resuspended in 10 μL of circular ligation reaction mix (1x CircLigase II buffer, 2.5 mM MnCl2, 50 U CircLigase II (Lucigen)). Circular ligation was performed at +60°C for 2 hours and the enzyme was inactivated through heating at +80°C for 10 min. To amplify the libraries the PCR reactions were performed with NEB Phusion polymerase and index primers (NEB, E6609S). The reaction mix of 50 µl (1x High fidelity buffer, dNTPs 0.2 mM each, 500 nM NEB Universal primer, 500 nM NEB Index primer, 2 µl circularization reaction, 1 U Phusion polymerase) was run according to the following program: 98 °C, 30 sec; 20 cycles of 98 °C, 10 sec; 65 °C, 10 sec; 72 °C, 10 sec; 72 °C, 5 min. Typically, the number of cycles was 16-18 for IGF2BP3 IP and 22-23 for IgG control. Libraries were ethanol precipitated, resolved on 10 well of 6% TBE gel similarly to the cycle selection PCR, and separated from the no-insert library and unreacted primers. Gel extraction was performed similarly to the extraction after reverse transcription reaction, but the gel pieces were not heated higher than +30 °C. After ethanol precipitation, pellets were resuspended in 10 µl of nuclease-free water. Library size distribution and quantity were determined using Agilent Bioanalyzer 2100 with High Sensitivity DNA Kit (Agilent) and via qPCR with NEBNext Library Quant Kit for Illumina (NEB). Libraries were sequenced on a NovaSeq 6000 S1 at SR100 mode (Illumina) at the Vienna BioCenter NGS facility.