title: GSM9478783: group2 MKRN2 KO SHSY5Y cells, Control; Homo sapiens; RIP-Seq
library_construction_protocol: The MKRN2 knock-out SHSY5Y cells transfected with Flag, CSDE1-Flag, and MKRN2-Myc plasmids were collected after 48hrs, and the cells pellet were resuspended with an approximately equal volume of polysome lysis buffer[100 mM KCl, 5 mM MgCl2, 10 mM HEPES(pH 7.0), 0.5% NP40, 1mM DTT, 100 units/ml RNase inhibitor (Vazyme), 400 μM vanadyl ribonucleoside complexes (VRC, NEB, USA) and Protease inhibitor cocktail(1:100, Roche)]. Incubated on ice for 10 min, centrifuged at 15 000 g for 15 min to clear lysate of large particles. anti-FLAG M2 Affinity gels (Sigma, USA) were washed with NT2 buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40 pH 7.4) before using for immunoprecipitation. 50μl of pre-cleared supernatant lysate were saved as the total cellular mRNA(input), and the rest of RNA-protein complexes were immunoprecipitated with anti-FLAG M2 Affinity gels (Sigma, USA) in 850μl of NT2 buffer supplemented with 100 units/ml RNase inhibitor, 400 μM VCR, 10μl of 100mM DTT and 20mM EDTA, and incubated for 4hours at 4℃ with tumbling end over end. Beads were washed with 1.0 ml of ice-cold NT2 buffer for five times supplemented with 0.5 M urea and 0.1% SDS, and then resuspended the beads in 100 μl of NT2 buffer supplemented with 30 μg of proteinase K (Roche) to release the RNP components. RNAs were extracted using Trizol (Invitrogen), and subjected to RIP-seq analysis. Strand-specific sequencing libraries were constructed using the Ribo-off rRNA Depletion Kit to remove ribosomal RNA, followed by the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina. The procedure included RNA fragmentation, reverse transcription, end repair, A-tailing, adapter ligation, and PCR amplification. The final libraries were quantified and sequenced on an Illumina NovaSeq 6000 platform.