title: GSM2192985: RYBPfl/fl.YAF2KO_RYBP_OHT_rep3; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were fixed for 1 hr in 2 mM EGS, followed by 15 min in 1% formaldehyde. Reactions were quenched by the addition of glycine to a final concentration of 125 µM. After cell lysis and chromatin extraction, chromatin was sonicated using a BioRuptor sonicator (Diagenode), followed by centrifugation at 16,000 x g for 20 min at 4°C, and the supernatant was taken for immunoprecipitation. Immunoprecipitations were performed overnight at 4°C using chromatin corresponding to 5 x 10^6 cells and were carried out in a total volume of 1 ml using approximately 3 µg of antibody. Antibody-bound chromatin was isolated on protein A agarose beads (RepliGen, Waltham, CA) or protein A magnetic Dynabeads (Invitrogen, Carlsbad, CA). Beads were washed with low salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP40, 1% Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)) and TE buffer (x2) (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). To prepare ChIP-seq material, ChIP DNA was eluted, and cross-links reversed at 65°C in the presence of 200 mM NaCl where required. Samples were then treated with RNase and proteinase K before being purified with ChIP DNA Clean & Concentrator™ kit (Zymo, Irvine, CA). Libraries were prepared using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® kit, with 8-10 PCR cycles. Libraries were purified using AMPure XP cleanup, and quantified by qPCR using KAPA Library Quantitation Standards (KAPA Biosystems). Libraries were sequenced as 40bp paired-end reads on Illumina NextSeq 500 platform.
attributes.tag: GEO Accession
attributes.value: GSM2192985