alias: GSM2467199
title: GSM2467199: Wild-type (m5C) Input2; Arabidopsis thaliana; MeDIP-Seq
library_construction_protocol: Plant samples were aliquot and total RNA was extracted using the RNeasy Plus Mini kit (QIAGEN) according to the manufacturer’s instructions and pooled together. RNA samples were fragmented into ~100-nucleotide-long fragments by RNA fragmentation buffer (100 mM Tris-HCl, pH 7.0, 100 mM ZnCl2). Fragmented RNA was incubated for 2 h at 4 °C with anti-m5C monoclonal antibody (Diagenode) in IP buffer (150 mM NaCl, 0.1% Igepal CA-630, 10 mM Tris-HCl, pH 7.4) supplemented with RNasin® Plus RNase inhibitor (Promega). The mixture was then immunoprecipitated by incubation with Protein A/G Plus-Agarose (Santa Cruz) that was pre-bound with bovine serum albumin (BSA) at 4 °C for an additional 2 h. After extensive washing, bound RNA was eluted from the beads with 6.7 mM 5-methylcytosine  (Sigma-Aldrich) in IP buffer, and precipitated by ethanol. NEBNext® UltraTM Directional RNA Library Prep Kit for illumina® (NEB) was used to construct the library from immunoprecipitated RNA and input RNA. Sequencing was done on Illumina Hiseq 2500 platform.
identifiers.namespace: GEO
identifiers.id: GSM2467199