title: GSM6423171: mES ATRXKOG34R H33Input; Mus musculus; ChIP-Seq
library_construction_protocol: 5 million cells were fixed in growth media with 0.4% formaldehyde for 10 min and quenched with 125 mM glycine. Chromatin was released by sequential lysis with cells lysis buffer (10 mM Tris pH 8, 10 mM NaCl, 0.2% Igepal) and nuclear lysis buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS). Chromatin was sheared to ≈300 bp with 12 rounds of sonication (30 s on, 30 s off) on a Bioruptor (Diagenode) and incubated overnight at 4 ᵒC with 5 μg H3.3 antibody (Abcam, Ab176840). Samples were immunoprecipitated with Protein A Agarose beads (Sigma-Aldrich, 05015979001) and washed sequentially with low salt (20 mM Tris pH 8, 2 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), high salt (20 mM Tris pH 8, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), and LiCl buffers (10 mM Tris pH 8, 1 mM EDTA, 0.25 M LiCl, 1% Igepal, 1% sodium deoxycholate). Chromatin was eluted with elution buffer (1% SDS, 100 mM NaHCO3), decrosslinked overnight at 65ᵒC, incubated with Proteinase K, phenol chloroform extracted and ethanol precipitated. For PML chromatin immunoprecipitation, cells were treated as above except that they were fixed with 2 mM EGS (Pierce 26103) for 45 min at room temperature in PBS. Formaldehyde was then added to 1% for 20 min and quenched with 125 mM glycine. Chromatin was sheared to under 500 bp with 20 rounds of sonication (30 s on, 30 s off) on a Bioruptor (Diagenode) and lysates were immunoprecipitated with 10 μg PML antibody (Millipore, mab3738). For the WT H3.3 R1 and H3.3 K27M H3.3 ChIP R1 samples: Ovation Ultralow System V2 using Nugen protocols M01379 v3.1. c-bot clustering using 200pM of library pool. Illumina Protocol 15006165 v02 Jan 2016. 75bp PE sequencing using Illumina HiSeq 3000. Illumina Protocol 15066493 Rev A, February 2015. For all other samples: Ovation Ultralow System V2 using Nugen protocols M01379 v3.1. Denaturing and on-board clustering using 2pM of library pool containing 1% PhiX, Illumina protocol 15048776 v15 Apr 2020. 75 SR sequencing using Illumina NextSeq550 High output mode and v2.5 chemistry. Illumina Protocol 15069765 v06, Jul 2019.