title: GSM3135674: placenta-input-4; Homo sapiens; RIP-Seq
library_construction_protocol: For RIP-seq, the tissues were isolated after a mechanical dissection, and all the samples were serially washed and immediately frozen in liquid nitrogen. Total RNA of tissue or cells was isolated using TRIzol and treated with DNase. RNA were then chemically fragmented, and subjected to immunoprecipitation with anti-m6A antibody. After stringent washing, bound RNA was eluted by competition with N6-methyladenosine, and used for library construction as IP samples. After removal of ribosomal RNA from total RNA, the input RNA samples was subsequently fragmented and subjected to library construction. For ChIP-seq, nine 10-cm dishes of pCDNA4-SFB-Tre-METTL4 stable transfected cells were treated by doxycycline (1 ng/μl) for 24 hr to trigger the expression of METTL3, then subject to 2-step fixation by 2 mM DSG (AppliChem) for 30 min and 1% formaldehyde for 10 min at room temperature16. The following processes were all carried out at 4°C unless otherwise emphasizednoted. After quenching with 125 mM glycine, the cells were harvested and lysed by 200 ul RIPA buffer (50 mM Tris-HCl, 1% NP40, 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, 150 mM NaCl) supplemented with 2 mM RVC, 1 mM DTT, 100 μM PMSF and protease inhibitor cocktail (Roche) per dish. The lysate were sonicated using Bioraptor (Diagenode) for 25 min (9 sec of sonication followed by 9 sec pause) and centrifuged for 10 min at 15000 rpm to remove insoluble matters. 100 μl lyaste was kept to serve as input, while the rest were was subject to streptavidin Sepharose (GE) binding for 2 hr. After washinged withby RIPA buffer for 3 times, and the bound protein-DNA complex were eluted by competition with 2 mg/ml biotin (Sigma) for 2 hr. Then the eluate were incubated with S-protein agarose (Millipore) overnight, washed withby RIPA for 3 times, digested with RNase A for 1 hr at 37°C to remove RNA, and at last eluted by proteinase K digestion at 56°C for 30 min. The DNA of input and IPed samples were was incubated at 56°C for 5 hr to reverse crosslink, and extracted by Phenol/Chloroform followed by ethanol precipitation. RIP-seq. The IP libraries were prepared using NEBNext Ultra RNA Library Prep Kit for Illumina  and the input libraries were prepared using NEBNext Ultra Directional RNA Library Prep Kit for Illumina following manufacturer's recommendations. For ChIP-seq samples, the libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina.
attributes.tag: GEO Accession
attributes.value: GSM3135674