alias: GSM1919512
title: GSM1919512: siYT521 Nucleus; Homo sapiens; RNA-Seq
library_construction_protocol: PAR-CLIP was performed as previously described (Xu et al., 2014). Breifly, Flag-tagged YTHDC1 was immunoprecipitated from transfected HeLa cells and treated with Rnase. Bound RNA fragments were isolated with TriZol following Proteinase K digestion and subject to ibrary construction. RIP was performed according to literature procedure (Peritz et al., 2006) using polyA selected RNA and ribosomal RNA depleted RNA as biological replicates. HeLa cells were fractionated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to manufacturer protocol with the following modifications: following cytoplasmic isolation, nuclei were washed extensively (4 x 200 μL) with PBS. RNA from each portion was collected using Directzol RNA miniprep (Zymo Research) and treated with DNaseI. Samples were depleted of ribosomal RNA prior to sequencing. mESC mRNA was isolated using TriZol reagent and purified using poly-T oligo magnetic beads. PAR-CLIP libraries were constructed using Illumina TruSeq Small RNA (Rep 1) or NEBNext Small RNA (Rep 2) library construction kits. RIP-seq and subcellular RNA-seq libraries were constructed using the Illumina TruSeq Stranded mRNA kit. mESC mRNA libraries were constructed using the NEBNext Ultra Directional RNA library construction kit.
identifiers.namespace: GEO
identifiers.id: GSM1919512