HITS-CLIP experiments using mouse brain

Two independent Csde1 HITS-CLIP experiments were performed with the mixture of three whole-brain tissue lysates of 15-day-old (P15) mice using the standard protocols. Tissues were triple washed with ice-cold phosphate-buffered saline (PBS), and ultraviolet (UV) cross-linking was performed with UV irradiation type C (254 nm) at 400 mJ/cm2. Samples were ground, and cell lysis was performed in cold lysis buffer (1× PBS, 0.1% SDS, 0.5% NP-40, and 0.5% sodium deoxycholate) supplemented with a 1% ribonuclease inhibitor (Takara, Kusatsu, Shiga, Japan) and 2% protease inhibitor cocktail (Roche, Basel, Switzerland) for 30 min. Cell lysates were cleared by centrifugation at 10,000 rpm for 10 min at 4°C, and the supernatants were used for immunoprecipitation. Study protocols comply with all relevant ethical regulations and were approved by the IRB of Central South University. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Central South University.

For DNA digestion, RQ1 (Promega, Madison, WI; final concentration, 0.05 U/μl) was added to the lysate and incubated at 37°C for 3 min. RNA digestion was performed by adding micrococcal nuclease (1:10,000, Thermo Fisher Scientific, Waltham, MA), followed by incubation at 37°C for 10 min. For immunoprecipitation, 600 μl of lysate was incubated with 15 μg of antibody or control immunoglobulin G (IgG) antibody overnight at 4°C. The immunoprecipitates were further incubated with protein A/G Dynabeads for 2 to 3 hours at 4°C. After applying to a magnet and removing the supernatants, the beads were sequentially washed with wash buffer (1× PBS, 1% SDS, 0.5% NP-40, and 5% sodium deoxycholate), high-salt wash buffer (5× PBS, 1% SDS, 0.5% NP-40, and 5% sodium deoxycholate), and PNK (polynucleotide kinase) buffer [50 mM tris (pH 7.4), 10 mM MgCl2, and 0.5% NP-40] twice.

After washing with PNK buffer as described above, dephosphorylation and phosphorylation were performed with FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific, Waltham, MA) and T4 polynucleotide kinase (Thermo Fisher Scientific, Waltham, MA). The immunoprecipitated protein-RNA complex was eluted from the beads by heat denaturing and resolved on a Novex 4-12% Bis-Tris precast polyacrylamide gel (Invitrogen, Carlsbad, CA). The protein-RNA complexes were cut from the gel (Invitrogen, Carlsbad, CA), and RNA was extracted with TRIzol after digesting the proteins. The recovered RNA was used to generate libraries with a TruSeq small RNA library preparation kit (Gnomegen, San Diego, CA) following the manufacturer’s instructions. Libraries corresponding to 150 to 250 base pairs (bp) were purified and quantified. The libraries were sequenced on the Illumina NextSeq 500 system with 151-bp paired-end reads by ABlife Inc. (Wuhan, China).

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