title: GSM2357607: Ewing sacroma patient 120 H3K36me3 1; Homo sapiens; ChIP-Seq
library_construction_protocol: DNA was isolated from 10 to 25 mg of snap-frozen tumors, cell lines, and MSCs by standard proteinase K digestion and phenol/chloroform extraction. DNA was quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Q32866) and the Qubit dsDNA BR Assay Kit (ThermoFisher Scientific, Q32850). ChIP-seq for three primary human tumors was done as described previously (Tomazou EM., et al, Cell Reports, 2015). Chromatin was prepared from 20 to 50 sections (25µm each) of snap-frozen tumors obtained by microtome sectioning. The following antibodies were used: H3K4me3 (Diagenode, C15410003-50, pAb-003-050), H3K27me3 (Diagenode, C15410195, pAb-195-050), H3K4me1 (Diagenode, pAb-194-050), H3K27ac (Diagenode, pAb-196-050), H3K56ac (Active Motif, 39281), H3K9me3 (Diagenode, pAb-193- 050), H3K36me3 (Diagenode, pAb-192-050). Library preparation for ChIP DNA and input control DNA was performed using the NEBNext Ultra kit (New England Biolabs, E7370S/L) following the manufacturer’s instructions. Quality control for the final libraries was done by measuring the DNA concentration with the Qubit dsDNA HS assay (Life Technologies, Q32851) on Qubit 2.0 Fluorometer (Life Technologies, Q32866) and by determining library fragment sizes with the Experion DNA 1K Analysis kit (Bio-Rad, 700-7107) on the Experion Automated Electrophoresis Station (Bio-Rad, 701-7000) For reduced representation Bisulfite-seq (RRBS), 100 ng of genomic DNA was digested for 12 hr at 37°C with 20 units of MspI (New England Biolabs, R0106L) in 30 μl of 1× NEB buffer 2. To retain even the smallest fragments and to minimize the loss of material, end preparation and adaptor ligation were performed in a single-tube setup. End fill-in and A-tailing were performed by addition of Klenow Fragment 3′ > 5′ exo- (New England Biolabs, M0212L) and dNTP mix (10 mM dATP, 1 mM dCTP, 1 mM dGTP). After ligation to methylated Illumina TruSeq LT v2 adaptors using Quick Ligase (New England Biolabs, M2200L), the libraries were size selected by performing a 0.75× cleanup with AMPure XP beads (Beckman Coulter, A63881). The libraries were pooled in combinations of six based on qPCR data and subjected to bisulfite conversion using the EZ DNA Methylation Direct Kit (Zymo Research, D5020) with the following changes to the manufacturer’s protocol: conversion reagent was used at 0.9× concentration, incubation performed for 20 cycles of 1 min at 95°C, 10 min at 60°C, and the desulphonation time was extended to 30 min. These changes increase the number of CpG dinucleotides covered by reducing double-strand break formation in larger library fragments. Bisulfite-converted libraries were enriched using PfuTurbo Cx Hotstart DNA Polymerase (Agilent, 600412). The minimum number of enrichment cycles was estimated by qPCR. After a 2× AMPure XP cleanup, quality control was performed using the Qubit dsDNA HS (Life Technologies, Q32854) and Experion DNA 1k assays (BioRad, 700-7107).
attributes.tag: GEO Accession
attributes.value: GSM2357607