title: GSM3754740: pTATA-loz1GFP_PlusZn_input1; Schizosaccharomyces pombe; ChIP-Seq
library_construction_protocol: Cells were then washed, resuspended in the lysis buffer (50mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate, 0.4mM DTT, and protease inhibitors) and lysed by vortexing for 7 x 1 min in the presence of zirconium beads.  To obtain cross-linked chromatin, cell lysates were centrifuged at 14,000 rpm for 5 min and the supernatant discarded.  After centrifugation, cross-linked chromatin was resuspended in lysis buffer, DNA sheared to an average length of 500 bp by sonication using Covaris Evolution, and cell debris removed by centrifugation at 14000 rpm for 5 min.   For immunoprecipitations, chromatin solutions were incubated with anti-GFP antibodies (Abcam Ab290) and protein A magnetic beads (Invitrogen™ Dynabeads™ Protein A) overnight at 4°C.   After Immuno-complexes were harvested the crosslinks were reversed by heating at 65°C for 5-7 hrs. The ChIP-seq library preparation and sequencing were performed by the Institute for Genomic Medicine at Nationwide Children's Hospital. ChIP-seq libraries were constructed from 5 ng of fragmented ChIP DNA using NEB Ultra II FS library prep kit (New England Biolabs, Ipswhich MA).  Briefly, DNA fragment ends were 5´ Phosphorylated, dA-tailed, and ligated with a unique, dual UMI indexed adaptor (Integrated DNA Technologies, Iowa). Following purification, using a magnetic-bead based approach (AMPure XP System), adaptor-ligated DNA was amplified by 8-9 PCR cycles.  Quality of libraries were determined via Agilent 2200 TapeStation using High Sensitivity D1000 reagents, and quantified using Kappa SYBR®Fast qPCR kit (KAPA Biosystems, Inc, MA).
attributes.tag: GEO Accession
attributes.value: GSM3754740