title: GSM4672944: Input_ALKBH5_Rep3; Homo sapiens; RIP-Seq
library_construction_protocol: Total RNA was isolated from scrambled and ALKBH5-siRNA transfected 143B cells using Trizol (Thermo 15596018) followed by mRNA isolation using a Dynabeast Direct Kit (Life Tech 61012). 2 μg of polyadenylated mRNA was diluted to 100 ml in water and fragmented to 100 to 200 nucleotides using Bioruptor (Diagenode). 5 ml of RNA was used for input preparation. 2 μg of fragmented mRNA was used for immunoprecipitation (IP) using rabbit anti-m6A antibody (Synaptic Systems #202003) in IP buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.1% NP-40) for 2 hours at 4°C. After incubation, samples were added to Protein A beads and incubated for 2 more hours at 4°C with rotation. After washing, samples were eluted using elution buffer containing m6A nucleoside and elute was concentrated using a Zymo RCC-5 column. The resultant final product was used for RNA-seq. 0.1 μg of fragmented mRNA was used as input control for RNA-seq. For replicate 3: same protocol was followed with another m6A antibodiy (ABE572, Millipore, Germany).  .For rep2, meRIP-seq was done with total RNA using the Magna MeRIP m6A kit (catalog no.17-10499, Millipore) according to the manufacture's protocol and it employed  MABE1006 (Millipore-Sigma) m6a antibody for IP. RNA-seq library preparation by following the Illumina TruSeq stranded mRNA sample preparation guide
identifiers.namespace: GEO
identifiers.id: GSM4672944