title: GSM8602283: SAFA_MinusTag_CLAP; Homo sapiens; Mus; RNA-Seq
library_construction_protocol: Cells were lysed in 1 mL lysis buffer (50mM Tris pH 7.5, 100mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1X Promega PIC). For CLIP samples, RNA was digested with Ambion RNase I (1:3,000 dilution) to achieve a size range of 100-500 nucleotides in length. We used a fixed RNase condition for all experiments to enable comparison between proteins. Lysates were precleared by mixing with Protein G beads (Dynabeads) for 30 minutes at 4°C. Target proteins were immunoprecipitated overnight at 4°C from 10 million cells with 5 μg of antibody coupled to 40 μL of Protein G beads in 100 μL lysis buffer. Antibodies were pre-coupled to beads for 1 hour at room temperature with mixing, followed by 3 washes with lysis buffer to remove unbound protein. After immunoprecipitation, beads were washed four times with high salt wash buffer (50 mM TrisHCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) and four times with wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20). RNA and protein were eluted by incubating in NLS elution buffer (20 mM Tris-HCl pH 7.5, 10 mM EDTA, 2% N-lauroylsacrosine, 2.5 mM TCEP) supplemented with 100 mM DTT at 50°C for 20 minutes. Samples were then run through an SDS-PAGE gel and transferred to a nitrocellulose membrane using the iBlot transfer system. The region 70 kDa above the molecular weight of the protein of interest was isolated and treated with Proteinase K (NEB), followed by purification with RNA Clean & Concentrate-5 (Zymo). Anti-V5 antibody (Bethyl, A190-120A) was used for all CLIP experiments.  For CLAP samples, cells were resuspended in 1 mL of cold lysis buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with 1X Protease Inhibitor Cocktail (Promega), 200 U of RiboLock (NEB), 20 U of TURBO DNase (Ambion), and 1X manganese/calcium mix (0.5mM CaCl2, 2.5 mM MnCl2). Samples were incubated on ice for 10 minutes and then at 37°C for 10 minutes at 1150 rpm shaking on a ThermoMixer (Eppendorf). Lysates were cleared by centrifugation at 15000g for 2 minutes and the supernatant was collected for capture to HaloLink Resin (Promega). For each CLAP capture, 200 μL of 25% HaloLink Resin (50 μL of HaloLink Resin total) was used per 10 million cells. Resin was washed three times with 2 mL of 1X TBS (50 mM Tris pH 7.5, 150 mM NaCl) and incubated in 1X Blocking Buffer (50 mM HEPES, pH 7.5, 10 μg/mL Random 9-mer, 100 μg/mL BSA) for 20 minutes at room temperature with continuous rotation. After the incubation, resin was washed three times with 1X TBS. Cleared lysate was mixed with 50 μL of HaloLink Resin and incubated at 4°C overnight with continuous rotation. The captured protein bound to resin was washed three times with lysis buffer at room temperature and then three times at 90°C for 3 minutes while shaking at 1200 rpm with each of the following buffers: 1X ProK/NLS buffer (50 mM HEPES, pH 7.5, 2% NLS, 10 mM EDTA, 0.1% NP-40, 10 mM DTT), high salt buffer (50 mM HEPES, pH 7.5, 10 mM EDTA, 0.1% NP-40, 1M NaCl), 8M urea buffer (50 mM HEPES, pH 7.5, 10 mM EDTA, 0.1% NP-40, 8 M Urea), and Tween buffer (50 mM HEPES, pH 7.5, 0.1% Tween 20, 10 mM EDTA). After the last wash, samples were centrifuged at 7500g for 30 seconds and supernatant was discarded. For elution, HaloLink Resin was resuspended in 100 μL of ProK/NLS buffer + 10 μL of Proteinase K (NEB) and incubated at 50°C for 20 minutes while shaking at 1200 rpm. Elutions were then transferred to microspin cups (Pierce, Thermo Fisher), centrifuged at 2000g for 30 seconds, and purified with RNA Clean and Concentrate-5 (Zymo).