Ena-DATASET-ErasmusMC-hema-19-12-2020-08:14:50:597-97 - samples
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ABSTRACT:
PROVIDER: EGAD00001006821 | EGA |
REPOSITORIES: EGA
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Project description:ChIP-seq was performed to assess changes in the activity of the MYC SE upon deletion of its modules or CTCF binding sites. Immunoprecipitation of crosslinked chromatin was performed with antibodies directed against H3K27Ac (Diagenode C15410196), H3K9Ac (Diagenode C15410004), H3K4me3 (Diagenode C15410003), RUNX1 (Abcam ab23980) or CTCF (Cell Signalling, 2899S). Crosslinks were reversed overnight at 65°C in the presence of proteinase K (New England Biolabs). De-crosslinked material was purified using a QIAGEN PCR Purification Kit. The purified DNA was processed according to the Nextflex ChIP Sample Preparation Protocol (Perkin Elmer) or the Microplex library preparation kit V2 (Diagnode C05010013) and sequenced on the Illumina NovaSeq6000 platform.
2021-07-26 | E-MTAB-9965 | biostudies-arrayexpress
Project description:Investigating how the Wnt-driven Mll1 epigenome regulates salivary gland and head and neck cancer. We performed mRNA-seq and ChIP-seq of H3K4me1, me2 and me3 on mouse salivary gland cancer cells that are kept in two different growth conditions, adherent culture and non-adherent sphere culture. Mouse salivary gland cancer cells were isolated from salivary gland of transgenic mouse that harbor K14-Cre-induced Wnt/β-catenin gain-of-function and Bmpr1a loss-of-function mutations. Anti-H3K4me1 (C15410194), -me2 (C15410035) and -me3 (C15410003-50) antibodies were purchased from Diagenode. ChIP-seq was performed according to the protocols provided by Diagenode using the iDeal ChIP-seq kit for histones and the iDeal library preparation kit. For mRNA-seq, mRNA was extracted according to the standard TRIzol protocol (Invitrogen) and subjected to library preparation using the TruSeq stranded mRNA library preparation kit. Sequencing was performed with the TruSeq SBS Kit v3-HS (2 X 200 cycles) on an Illumina HiSeq 2000 sequencer.
2018-12-24 | E-MTAB-7514 | biostudies-arrayexpress
Project description:ChIP-Seq is a powerful method commonly used to study global protein-DNA interactions including both transcription factors and histone modifications. We have found that the choice of ChIP-Seq library preparation protocol plays an important role in overall ChIP-Seq data quality. However, very few studies have compared ChIP-Seq libraries prepared by different protocols using multiple targets and a broad range of input DNA levels. In this study, we evaluated the performance of four ChIP-Seq library preparation protocols [NEB NEBNext Ultra II, Roche KAPA HyperPrep, Diagenode MicroPlex, and Bioo (now PerkinElmer) NEXTflex] on three target proteins, chosen to represent the three typical signal enrichment patterns in ChIP-Seq experiments: sharp peaks (H3K4me3), broad domains (H3K27me3) and punctate peaks with a protein binding motif (CTCF). We also tested a broad range of different input DNA levels from 0.10 to 10 ng for H3K4me3 and H3K27me3 experiments. Our results suggest that the NEB protocol may be better for preparing H3K4me3 (and potentially other histone modifications with sharp peak enrichment) libraries; the Bioo protocol may be better for preparing H3K27me3 (and potentially other histone modifications with broad domain enrichment) libraries, and the Diagenode protocol may be better for preparing CTCF (and potentially other transcription factors with well-defined binding motifs) libraries. For ChIP-Seq experiments using novel targets without a known signal enrichment pattern, the NEB protocol might be the best choice as it performed well for each of the three targets we tested across a wide array of input DNA levels.
2023-06-23 | GSE196402 | GEO
Project description:RNAseq was done using Library protocol =Illumina TruSeq Stranded mRNA Library Preparation Kit with poly(A) selection , HiSeq 101 Cycle Paired-End Sequencing v4
2018-11-30 | GSE114575 | GEO
Project description:Total RNA was isolated from mouse Asxl2-/- and WT LK cells following standard protocol with TRIZol reagent (Life Technologies) followed by RNA library preparation with the Illumina TruSeq strand-specific mRNA sample preparation system. All RNA-seq libraries were sequenced with a read length of single-end 75bp using the Illumina NextSeq 500, and final of over 45 million reads per sample.
2018-03-08 | GSE97021 | GEO
Project description:The DNA methylome of 45 primary neuroblastoma tumors is profiled by enrichment with a methyl-CpG-binding domain (MBD) and massively parallel sequencing DNA of 45 primary tumors is sheared (fragments of ± 200 bp), followed by MBD-based (MethylCap kit of Diagenode) enrichement, library preparation and multiplexing. Both input DNA and captured DNA were sequenced paired-end on Illumina Hiseq2000
2015-11-19 | E-GEOD-69243 | biostudies-arrayexpress
Project description:The DNA methylome of 15 primary stage 4S neuroblastoma tumors is profiled by enrichment with a methyl-CpG-binding domain (MBD) and massively parallel sequencing DNA of 15 primary stage 4S tumors is sheared (fragments of ± 200 bp), followed by MBD-based (MethylCap kit of Diagenode) enrichement, library preparation and multiplexing. Both input DNA and captured DNA were sequenced paired-end on Illumina Hiseq2000
2015-12-15 | E-GEOD-69268 | biostudies-arrayexpress
Project description:ChIP-seq data (H3K4Me1, H3K4Me3, H3K27Ac histone modifications) in experimental triplicates on multiple myeloma cell line KMS11 and plasma cell leukaemia cell lines L363 and JJN3. ChIP reactions were performed on a Diagenode SX-8G IP-Star Compact using Diagenode automated Ideal Kit. ChIP libraries were generated using HTP Illumina library preparation kit, and sequenced using Illumina HiSeq 2000 with 100 bp single-ended reads. ChIP-seq files are in BED format.
| EGAD00001003349 | EGA
Project description:U2OS osteosarcoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) and Raji B-cells in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS, Bio&Sell), 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco) at 37°C at 8% or 5% CO2, respectively. Chromatin immunoprecipitation for ChIP-seq: Cells were crosslinked using a formaldehyde containing solution (10mM NaCl, 0,1mM EDTA pH 8, 0,05mM EGTA pH 8, 5mM HEPES pH 7.8 and 1% formaldehyde) for 10 minutes at 20°C, the reaction was quenched by the addition of glycin to a final concentration of 250uM for 5 minutes. Crosslinked cells were collected and washed twice with PBS before snap freezing in liquid nitrogen and storage at -80°C until subsequent use. Prior to sonication, the crosslinked cells were resuspended in lysis buffer (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100, 1X protease inhibitor cocktail) at 4°C for 20 minutes. Nuclei were collected by centrifugation and washed in a second buffer (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 1X protease inhibitor cocktail) for 10 minutes at 4°C then collected by centrifugation and resuspended in the shearing buffer (1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1X protease inhibitor cocktail). Sonication was carried out in a Bioruptor Pico ultrasounds water bath (Diagenode B01060001) for 30 cycles of 30 sec ON and 30 sec OFF pulses in 4°C water. Sonicated extracts were centrifuged at high speed in the presence of 0,1% of Triton X-100 and snap frozen in liquid nitrogen then stored at -80°C until subsequent use. Prior to ChIP, the ZNF768 mAb was coupled to protein-G coated magnetic beads (Dynabeads, life technologies) by incubation in 0,5% BSA PBS overnight at 4°C. Pre-coated beads were then washed and incubated with the sonicated chromatin extracts, the ChIP was carried out overnight at 4°C on a rotating wheel. The equivalent of 10 × 107 cells sonicated extract was used for each ZNF ChIP experiment for both cell lines. After incubation, the beads were washed 7 times with Wash buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate, 1X protease inhibitor cocktail) followed by one wash with TE-NaCl buffer (10mM Tris pH 8 and 1mM EDTA pH 8, 50mM NaCl). Immunoprecipitated chromatine was eluted by two sequential incubations with 100µl Elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) at 65°C for 15 minutes. The two eluates were pooled and incubated at 65°C for 12 hours to reverse-crosslink the chromatin followed by treatment with RNase A (0.2µg/mL) at 37°C for two hours and Proteinase K (0.2µg/mL) at 55°C for two hours. The DNA was isolated by phenol:chloroform: isoamylalcohol (25:24:1 pH 8) extranction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). At least 1ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol. The ChIP DNA was size selected using Ampure beads (Life technologies) to enrich for fragments < 400Bp prior to end-repair, 3’end adenylation and adapter ligation. Library fragments were then directly amplified by 10 cycles of PCR.
2018-10-29 | GSE111879 | GEO
Project description:DNA methylation profiles of the livers of 1 day old rats from mothers fed with three different diets during gestation. The first animal group was fed with a normal diet (c=control); second group received much less protein than normal and slighlty more carbs (p=low protein, or programmed); third group diet was same as low protein but with extra folic acid (f=low protein+folate). All diets were matched for energy. Genomic DNA from rat livers was subjected to selection according to the methyl-CpG binding domain-based (MBD) protein protocol (DNA pooled from 6 individuals per group) and the resulting fragments sequenced in high throughput. Methylated DNA was captured with MethylCap kit (Diagenode). Approximately 10 ng of captured DNA was used for library preparation (ChIP-seq DNA sample Prep Kit, Illumina).
2014-05-20 | E-GEOD-50935 | biostudies-arrayexpress