title: GSM2473979: 5mC_KO_2 MeDIP-seq; Mus musculus; MeDIP-Seq
library_construction_protocol: For ChIP, mouse ES cells (C57BL/6N x 129/Sv background) stably expressing FHA-Srap1 were fixed in 1% paraformaldehyde for 10 min at 37 °C, followed by quenching with 125 mM glycine for 5 min. Cells were lysed and processed using the MAGnify ChIP system (Life Sciences). Lysates were sonicated using an ultrasonic processor (Cole Parmer) at power setting 3 in 30 s pulses followed by 1 min on ice, for a total of 6 min sonication. Nucleoprotein complexes were immunoprecipitated using a mixture of M2 Flag and HA antisera (2.5 mg each) or equivalent amounts of isotype-matched mouse IgG per sample. For DIP experiments, genomic DNA was isolated from Srap1 KO and matched wild-type ES cells using the DNeasy Blood & Tissue DNA kit and sonicated for a total of 6 min. Samples were processed using the MeDIP or hMeDIP kits (Active Motif) according to the manufacturer’s protocol. For 5fC/5caC DIP, the hMeDIP protocol was followed using polyclonal antibodies to 5fC or 5caC. Library preparation was performed using the Kapa HyperPrep kit, followed by 12 cycles of PCR amplification with Kapa HiFi HotStart polymerase. Amplified libraries were pooled, purified twice with 0.9X Ampure XP beads and analyzed on the Illumina HiSeq2000 or NextSeq500 for single-end reads.
attributes.tag: GEO Accession
attributes.value: GSM2473979