title: GSM4411132: STO_DNAme_IP_rep2; Mus musculus; MeDIP-Seq
library_construction_protocol: For RIP-Seq,total RNA was extracted from FGSCs and STO using Trizol reagent (Life Technologies, CA). Next, 20ug of the digested RNA was used to puritying mRNA and the mRNA was used for the VAHTSTM mRNA-seqv2 Library Prep kit for Illumina1, and sequencing libraries were created according to the manufacturer's protocol. For ChIP-Seq, lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. For MeDIP-Seq, genomic DNA were extrated and sonicated,and the incubated the antibody. For RIP-Seq, Isolation of mRNA was performed using the Oligotex mRNA Mini Kit (Qiagen GmbH, Hilden, Germany) and the mRNA was then used for RIP-Seq library preparation with the NEB Next Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). The library was then subjected to Illumina sequencing with paired-end 2x150 as the sequencing mode.For ChIP-Seq and MeDIP-Seq,Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
attributes.tag: GEO Accession
attributes.value: GSM4411132