title: GSM9024485: Mouse uterus aged group3 rep2 H3K27ac; Mus musculus; ChIP-Seq
library_name: GSM9024485
library_construction_protocol: Ultra-low input native ChIP-seq was performed on pieces of snap frozen endometrial tissue as previously described [ref]. Briefly, tissue was thawed on ice in 100μL nuclear isolation buffer ﻿(NIB, Sigma) and diced using a blade. An aliquot of 20,000 nuclei in 10μL NIB was then taken forward for Mnase (NEB) digestion at 21 °C for 7.5 minutes. Digested chromatin was then suspended in Complete IP buffer ﻿(20mM Tris-HCl (pH8), 2mM EDTA, 150mM NaCl, 0.1%Triton-X100, 1x Protease inhibitor cocktail,1mM PMSF). Following pre-clearing the chromatin on a mix of protein A/protein G beads (Thermo Fisher Scientific), and after 10% was set aside for input, the chromatin was divided for immunioprecipitation overnight rotating at 30 rpm, at 4 °C. IP was performed using 250ng antibodies for H3K4me3 ﻿(Diagenode, C15410003) and H3K27ac ﻿(Abcam, ab4729). DNA was washed in low then high salt washes, before eluting ﻿in ChIP elution buffer (100mM NaHCO3, 1% SDS). DNA was then SPRI purified before continuing to library preparation Libraries were prepred using the ﻿MicroPlex Library Preparation kit (Diagenode, C05010001). Libraries were quantitated using the Agilent Bioanalyzer High Sensitivity DNA assay before pooling for sequencing. Libraries were sequenced on the Illumina NextSeq500, 75 bp paired end.