{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Patrick Nylund"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14627"],"description":["Multiple myeloma (MM) cell line treated with an inhibitor to EZH2/EZH1 (UNC1999), DNA methylation inhibitor (5-azacytidine), and a combination of both."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Growth Protocol - The MM cell line INA-6 was cultured in RPMI-1640 AQmediaTM media (Sigma) supplemented with 10% heat-inactivated FBS (Sigma), 1% GlutaMAXTM-I and antibiotics (penicillin 100U/ml and streptomycin 50 mg/mL; Sigma) and Interleukin-6 (IL-6) at 37°C in a humidified 5% CO2 in-air atmosphere incubator. Exponentially growing cells were seeded at 100.000 cells/mL and incubated overnight before addition of reagents. Every 72h, cells were split in equal numbers and re-plated.","Library Construction - SMARTer ThruPLEX DNA-seq library preparation kit WITH  fragmentation (Takara Bio).","Sequencing - Cluster generation and 50 cycles paired-end sequencing of  the libraries in 1 NovaSeq S2 flowcell using  the NovaSeq 6000 system and v1.5 sequencing chemistry (Illumina Inc.).","Sample Collection - ChIP was conducted by utilizing the iDeal ChIP-seq Kit for Histones (Diagenode, #C01010051) according to the manufacturer’s instructions. In short, 7 million INA-6 cells treated with vehicle DMSO, UNC1999, 5-azacytidine or combination, were cross-linked for 8 min at room temperature in 1% formaldehyde (Thermo Scientific, #28906). Then, 0.1 M of glycine was added to cease the cross-linking reaction. Shearing of chromatin was done in 10 cycles on the PicoBioruptorTM (Diagenode) (30 sec on/30 sec off). Samples were then incubated with either anti-H3K27me3 (5.68µg) (Diagenode, #C15410196), anti-H3K27ac (2µg) (Active Motif, #39155), anti-H3K4me1 (2µg) (Abcam, #8895) or anti-H3K4me3 (2µg) (Millipore, #07-473) antibodies.","Nucleic Acid Extraction - iDeal ChIP-seq Kit for Histones (Diagenode, #C01010051) according to the manufacturer’s instructions.","Sample Treatment - 7 million INA-6 cells treated with vehicle DMSO, 0.5µM UNC1999, 50nM 5-azacytidine or the combination of 0.5µM UNC1999 and 50nM 5-azacytidine for 9 days."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - The ChIP-Seq fastq files were processed using the nf-core (Ewels et al. 2020) ChiP-Seq pipeline in version 1.2.2 (doi:  10.5281/zenodo.3240506) and using default parameters for broad (H3K27me3, H3K4me1) and narrow (H3K4me3, H3K27ac) peaks.  A custom reference genome consisting of the concatenation of the GRCh38 human and Drosophila melanogaster (dm6) reference genome sequences was used.  Similarly, a custom blacklist BED file was generated by concatenating GRCh38 and dm6 blacklist regions.  Briefly, the nf-core pipeline performed the following steps:  trim adapters by Trim Galore!, map reads by BWA, filter reads, report QC measures for read alignment and ChIP, call peaks by MACS2, generate a consensus peak set per mark using BEDTools, count reads in consensus peak set by featureCounts.  The chromatin samples from the INA-6 cell line were exposed to four different treatments, namely DMSO, 5-azacytidine, UNC1999 and a combination of 5-azacytidine and UNC1999.  A constant small amount of sonicated dm6 chromatin spike-ins (33.25-36ng) was added before immunoprecipitation.  This was performed for four batches of replicates.  A scaling factor was then computed for each replicate batch using DESeq2 function estimateSizeFactorsForMatrix(Love, Huber and Anders 2014) using counts of dm6 peaks only.  All peak counts were subsequently divided by the dm6-derived batch-specific scaling factor to correct for technical variability in the ChIPSeq experiment.  To determine peaks that were statistically significant between UNC1999 and DMSO, we computed the fold change per batch and then performed a hypothesis test using a two-sided t-test on four values of fold changes, and under the null hypothesis that the mean is zero.  Finally, the false discovery rate to account for multiple testing was computed using the Benjamini-Hochberg method. Data was visualised with IGV V.2.11.0 (Robinson et al. 2011)."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["ChIP-seq"],"species":["Homo sapiens"],"pubmed_authors":["Antonia Kalushkova","Patrick Nylund"],"additional_accession":[]},"is_claimable":false,"name":"ChIP-seq INA-6","description":"Multiple myeloma (MM) cell line treated with an inhibitor to EZH2/EZH1 (UNC1999), DNA methylation inhibitor (5-azacytidine), and a combination of both.","dates":{"release":"2025-12-29T00:00:00Z","modification":"2025-12-29T02:02:00.832Z","creation":"2024-11-22T14:10:35.655Z"},"accession":"E-MTAB-14627","cross_references":{"ENA":["ERP166445"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0002692","EFO_0004917","EFO_0005518","EFO_0004184","EFO_0003969"]}}