GSM3024913: PDX DMSO H3K27ac; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Unclassified
Antigen
Unclassified
Cell type
Cell type Class
Blood
Cell type
Acute myeloid leukemia
NA
NA
Attributes by original data submitter
Sample
source_name
PDX2 patient derived xenograft
tissue
PDX2 patient derived xenograft
cell type
AML
treatment
0.05% DMSO
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were washed once with PBS and fixed with 1% paraformaldehyde for 10min. Glycine was added at a final concentration of 0.125M to stop the reaction. Cells were collected at 500 g for 10 min at 4 °C (subsequent work was performed on ice and used cool buffers and solutions unless otherwise specified) and washed twice with up to 0.5 ml ice-cold PBS supplemented with 1X Xpert Protease Inhibitor Cocktail Solution. Cells were pelleted, flash frozen and stored at -80C or used immediately for downstream applications. ChIPmentation was carried out as previously described (Schmidl et al., 2015), with minor adaptions. Cell pellets were lysed in sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.25% SDS, 1X protease inhibitors) and sonicated with a Covaris S220 sonicator for 9 min in a microTUBE until the size of fragments was in the range of 200–700 bp. Lysates were transferred to new tube and diluted 1:1.5 with equilibration buffer (10mM Tris, 233mM NaCl, 1.66% Triton X-100, 0.166% sodium deoxycholate, 1 mM EDTA, 1X protease inhibitors). Lysates were centrifuged at full speed for 10 min at 4 °C and the supernatant containing the sonicated chromatin was transferred to a new tube. The lysate was then brought to RIPA buffer conditions (final concentration: 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1X protease inhibitors) to a volume of 100 μl per immunoprecipitation. The antibody was added to the lysates and incubated on a rotator overnight at 4 °C. Used antibodies were H3K4me3 (1 μg per immunoprecipitation, Cell Signaling clone C42D8), H3K27ac (1 μg per immunoprecipitation, Cell Signaling clone D5E4) and H3K27me3 (1 μg per immunoprecipitation, Cell Signaling clone C36B11). 5μl magnetic Protein A beads (Life Technologies) were washed twice and resuspended in 10ul of RIPA-LS supplemented with 0.1% BSA. Blocked beads were added to immunopreciptated lysates and incubated for 2 hours on a rotator at 4 °C. Beads were washed subsequently with RIPA-LS (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate and 1X protease inhibitors) (twice), RIPA-HS (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate and 1X protease inhibitors) (twice) and RIPA-LiCl (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.5% NP40 and 1X protease inhibitors) (twice). Beads were washed with cold Tris-Cl pH 8.0, to remove detergent, salts and EDTA. Beads were washed once more with cold Tris-Cl pH 8.0 but the reaction was not placed on a magnet to discard supernatant immediately. Instead, the whole reaction including beads was transferred to a new tube and then placed on a magnet to remove supernatant to decrease background. Beads were then resuspended in 25 μl of the tagmentation reaction mix (10 mM Tris pH 8.0, 5 mM MgCl2, 10% v/v dimethylformamide) containing 1 μl Tagment DNA Enzyme from the Nextera DNA Sample Prep Kit and incubated at 37 °C for 2 min in a thermocycler. The beads were washed with RIPA-LS (twice) and once with cold Tris-HCl pH 8. Beads were washed once more with cold Tris-HCl pH 8.0 but the reaction was not placed on a magnet to discard supernatant immediately. Instead, the whole reaction including beads was again transferred to a new tube and then placed on a magnet to remove supernatant. Beads were then incubated with 50 μl elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA and 10 mM Tris-HCl pH 8.0) containing 2 μl of Proteinase K for 1 h at 55 °C and 8 h at 65 °C, to revert formaldehyde cross-linking, and supernatant was transferred to a new tube. Finally, DNA was purified with Qiagen MinElute columns and eluted in 22ul of elution buffer (10mM Tris-HCl pH 8.0). Enrichment of the libraries was performed in a 50-μl reaction using 0.75 μM primers, 25μl NEBNext High-Fidelity 2X PCR Master Mix and 20ul of the purified library. Libraries were amplified for N+1 cycles, where N is equal to the rounded-up Cq value determined in a test qPCR reaction with 1ul of the library. Enriched libraries were purified using SPRI AMPure XP beads at a beads-to-sample ratio of 1:1, followed by a size selection using AMPure XP beads to recover libraries with a fragment length of 200–400 bp. Library preparation was performed using custom Nextera primers as described for ATAC-seq (Buenrostro et al., 2013). ChIP-seq libraries were run on an Illumina NextSeq 500 instrument (single end 75 bp reads).