Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Briefly, cells were crosslinked for 5 min at room temperature with 1% fresh formaldehyde. Reaction was quenched with glycine (125 mM final concentration) for 5 min at room temperature. Cells were lysed in 0.1% SDS lysis buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0,150 mM NaCl). Chromatin was sonicated to a mean size of 300-500bp. Lysed cells were cleared by centrifugation and anti-Ring1b (Active Motif 39664) was added to chromatin extracts and incubated overnight. Protein A/G Dynal beads were added for 2 hours and beads were isolated with a magnet and then washed with 0.1% SDS Lysis Buffer, Low Salt Buffer ( 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), LiCl Wash Buffer (250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0), and TE x2. DNA was eluted and decrosslinked overnight at 65 oC in SDS Elution Buffer (1% SDS, 10mM EDTA, 50 mM Tris-HCl pH 8.0). The next day RNase A and proteinase K were added, and the DNA was precipitated in the presence of glycogen. DNA for downstream applications were quantitated by flourometry (Qubit, Invitrogen), and equal DNA was used for each ChIP-qPCR and normalized to sheared, non-IP’d genomic DNA (Input). NEB ChIp-seq library prep kit (cat# E7645)