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Sample GSM3597418

Query DataSets for GSM3597418

Status

Public on Jul 19, 2019

Title

ChIP-seq-FL-Ebf1-KO-a-RUNX1-ChIP

Sample type

SRA

Source name

Ebf1-/- FL

Organism

Mus musculus

Characteristics

strain background: C57BL6/J
genotype/variation: Ebf1 knock out
cell type: ProB
cuture protocol: Cultured in Optimem +10percent FBS, 25mM HEPES, 50microg/ml Gentamicin, 50microM beta-mercaptoethanol FL, KL, IL7 on OP9
antibody: Rabbit polyclonal anti-RUNX1 [ab23980, Abcam]

Treatment protocol

Cell culture: Fetal liver cells were purified from wild-type, Ebf1 deficient, or Pax5 deficient fetus, and expanded on OP9 stromal cells in Opti-MEM supplemented with 10percent heat-inactivated fetal calf serum, 25mM HEPES, 50μg/ml Gentamicin, 50μM β-mercaptoethanol, 10ng/ml KIT ligand, 10ng/ml Fms-like tyrosine kinase 3 ligand, and 10ng/ml Interleukin-7. Murine pre-B cell line, 230-238 cells were maintained in RPMI1640 supplimented with 10percent heat-inactivated fetal calf serum, 25mM HEPES, 50μg/ml Gentamicin, and 50μM β-mercaptoethanol. Infection: Retronectin coated 24-well plates were pre-inkubated with 500ul viral supernatant. Cells were harvested, resuspended 500ul media and added to 500μl of virus supernatant. Polybrene was added to supernatant at concentration of 0.5μg/ml. Spin-infection was performed at 1800xg, for 90min at 32°C.
230-238-cells were cultured in RPMI1640 with UltraGlutamine (Lonza, Basel, Swiss) supplemented with 10percent heat-inactivated FCS, 20mM HEPES, 50mg/ml Gentamicin and 50mM β-ME.

Extracted molecule

genomic DNA

Extraction protocol

For RNA-seq: Total RNA was isolated by use of RNAeasy Micro Kit (Qiagen, Hilden, Germany) according to manufacturer’s recommendations. For ChIP-seq: Cells for transcription factor ChIP were fixed at room temperature with DSG for 30 min followed by 1percent formaldehyde/PBS for 10 minutes. Reaction was then quenched by adding glycine to 0.125 M. Cells were washed pelleted and snap-frozen and stored at -80°C until ready for ChIP or used immediately for ChIP. Nuclei for ChIP were isolated by 10 min incubation in Nuclei Isolation buffer (50 mM Tris-pH 8.0, 60 mM KCl, 0.5percent NP40) + protease inhibitor cocktail (PIC) (Roche) on ice. Pelleted nuclei were dissolved in Lysis buffer (0.5percent SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8))+ PIC and sonicated on a Bioruptor (Diagenode) max power for 30s followed by 30 s rest. Sonication was followed by pelleting of debris and the supernatant was transferred to new tube and chromatin was diluted 5X in Dilution Buffer (1percent Triton, 2mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8) + PIC). Anti-bodies were hybridized to ProteinA or G Dynabeads® (LifeTechnologies) and added to the diluted chromatin. ChIP was performed over night at 4°C. and subsequently washed (1 time with 500 μl Low Salt Immune Complex Wash Buffer, 1 time with 200 μl High Salt Immune Complex Wash Buffer, 1 time with 200 μl LiCl Immune Complex Wash Buffer, 2 times with 200 μl TE buffer) and eluted for 4 h at 65°C ( 20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1percent SDS, 100 μg RNase A and 50 μg proteinase K) treated and finally cleaned up using Zymo ChIP DNA Clean & Concentrator before ChIP-seq libary preparation.
RNA-seq: Libraries were constructed using NuGEN’s Ovation Ultralow Library systems (NuGEN Technologies, San Calros, CA) and were subsequently subjected to 76 cycles of NextSeq500 sequencing (Illumina, San Diego,CA). ChIP-Seq: Libraries were constructed using Illumina Truseq Nano DNA library preparation kit or by addition of Bioo-scientiffic Nextflex barcodes following standard procedures. Libraries were run on the Illumina NexSeq500.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Description

Peaks-FL-EKO-a-RUNX1-vs-input

Data processing

RNA-Sequencing and data analysis: Libraries were single-end sequenced on a NexSeq500. For analysis of RNA-Seq experiments the reads were aligned to mouse reference genome (mm10 / GRCm38) using STAR (2.6.0b-1) (Dobin, A etal. 2012),) with single read, standard settings. If not indicated, further analyses were performed using the HOMER platform (v4.8, v4.10) (Heinz et al. 2010). For analysis of statistically significance among differently expressed genes, the data was analyzed using analyzeRepeats.pl with the – noadj option, followed by the getDiffExpression.pl command using edgeR(3.12.0).
ChIP-sequencing and data analysis: For ChIP-Seq samples, Libraries were single-end sequenced on a NexSeq500, reads were aligned to the mm10 genome using default single end parameters for Bowtie2 (2.3.4.2) (Langmead, B. etal. 209 and 2012). Aligned read files from ChIP seq experiments were analyzed with HOMER (v4.8, v4.10) (Heinz et al. 2010) to find peaks, perform motif-enrichment, and other analyses in the study.
Genome_build: mm10
Supplementary_files_format_and_content: peak-files, annotated peak-files and expressionmatrices in txt format

Submission date

Feb 11, 2019

Last update date

Jul 19, 2019

Contact name

Mikael Sigvardsson

E-mail(s)

[email protected]

Organization name

Lund University

Lab

Sigvardsson