title: GSM4550956: GFP-DNMT1-dCAT; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP sequencing, 293T cells were plated on a 6-well dish and, when they reached at 70~80% confluency, 2 µg of pEGFP-Dnmt1s, pEGFP-Dnmt1sc-, or pEGFP-c2 (Clontech) plasmid was transfected into the cells using Lipofectamine LTX (Thermo) according to manufacturer's protocol. Cells were harvested 72 hrs after transfection and 1 x 106 cells were used in chromatin immunoprecipitation (ChIP) as described previously. Briefly, for cross-linking of chromatin and proteins, cells were incubated in 1% formaldehyde solution for 10 min at RT, neutralized by glycine treatment, and lysed to extract chromatin. The resulting chromatin was sheared to the size ranging from 200 - 500 bp, reacted with 2 µg a-GFP antibodies (Santa Cruz) or IgG control (Cell Signaling), and captured by Protein A magnetic beads (Bio-Rad). After reverse cross-linking and purification, the eluted DNA fragments were proceeded for Illumina sequencing library generation. ChIP-seq libraries were prepared using NEBNext DNA Library Prep modules (NEB) and NEBNext Multiplex Oligos (NEB) according to manufacturer's protocol, and paired-end sequencing data were generated by NEXTseq 550 (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4550956