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Sample GSM2410027

Query DataSets for GSM2410027

Status

Public on Dec 20, 2016

Title

BM-LSK-cells-undifferentiated [ATAC-seq-LSK_1]

Sample type

SRA

Source name

Wt-BM-LSK-cells

Organism

Mus musculus

Characteristics

strain: C57BL6/J
treatment: Fresh-sorted

Extracted molecule

genomic DNA

Extraction protocol

Cell culture: Normal lineage negative Sca1+Kit+ (LSK) cells or lymph node pro-B cells (Lin- (CD11b, Gr1, Ter119 and CD11c) NK1.1-IgM-CD3-CD19+CD43+ were sorted and expanded in vitro by co-culture on OP9 stroma cells using OptiMEM supplemented with 10% 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 (FLT3L), and 10ng/ml Interleukin-7. For constitutively active Notch1 expression experiments, ICN1-pMIG or pMIG (control) retrovirus (MSCV) transduced cells were differentiated on OP9 for 14 days. For extra-cellular Notch experiment control cells were co-cultured on OP9 or OP9-DL1 stroma cells for 14 days. Both for extracellular Notch and the constitutive Notch1 signal experiments, the above-mentioned media and the cytokines were used. During C/EBPα transduction experiment C/EBPα-ER-pMIG or pMIG (Control) retrovirus were transduced into pro-B cells and cultured either with or without 4-HydroxyTamoxifen (1μM; Sigma; H7904) supplemented with 10% heat inactivated Charcoal stripped fetal calf serum, 25mM HEPES, 50μg/ml Gentamicin, 50μM β-mercaptoethanol, 10ng/mL Fms-like tyrosine kinase 3 ligand (FLT3L), 10ng/ml Interleukin-7, 10ng/ml IL3, 10ng/ml SCF 10ng/mL M-CSF and analysed by flow cytometry and the cells were sorted for RNA-Seq or ATAC-Seq. FACS staining. Frozen lymph node cells were CD16/CD32 (Fc)–blocked, stained and sorted based on the following markers (Lin- (CD11b,Gr1,Ter119 and CD11c) NK1.1-CD3-CD19+CD43+ and expanded in vitro by co-culture on OP9 stroma cells. For in vitro differentiation (pro-B to T cells) experiments, CD16/CD32 (Fc)–blocked cells were stained with antibodies against CD3, CD19 and Thy1.2. For in vitro (pro-B to Myleoid) differentiation analysis CD16/CD32 (Fc)–blocked cells were stained with antibodies against CD11b/Mac1 and CD19. Analysis and cell sorting was performed on a BD FACSAriaTM (BD Biosciences, San Jose, California) using propidium iodide (PI, In vitrogen, Paisly UK) as viability marker.
For RNA-seq: Total RNA was isolated by use of RNAeasy Micro Kit (Qiagen, Hilden, Germany) according to manufacturer’s recommendations. For ATAC-seq: Cells were washed in ice cold PBS prior to Assay for Transposase Accessible Chromatin (ATAC-seq) library preparation as described in (Buenrostro et al. 2013).
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). ATAC-seq: ATAC-seq library preparation was done as described in (Buenrostro et al. 2013) using Transposomes from Nextera DNA library preparation kit (Illumina, San Diego) and custom primers.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Data processing

RNA-Sequencing and data analysis: Each biological sample was processed and sequenced in duplicate or triplicate. For analysis of RNA-Seq experiments the reads were aligned to mouse reference genome (mm10 / GRCm38) using TopHat. If not indicated, further analyses were performed using the HOMER platform (Heinz et al. 2010). For further analyses we used normalization to 10M mapped reads by the analyzeRepeats.pl command with the option –count exons condeseGenes. For analysis of statistically significance among differently expressed genes, the data was analyzed using analyzeRepeats.pl with the – noadj option, and filtered by ≥ 50 tags repeats followed by the getDiffExpression.pl command using edgeR. Data normalized to 10M mapped reads per experiment in log scale was visualized by Java Treeview following hierarchical clustering in Cluster3 (centering genes on mean, average linkage cluster). BED files were created from HOMER tagdirectories.
ATAC-Sequencing and data analysis:Libraries were single-end sequenced on a NexSeq500. Each biological sample was processed for ATAC-Seq and sequenced in triplicate. The data was mapped to mm10 using Bowtie2 with standard settings. Tag directories with reads mapped to the mitochondrial chromosome filtered out were created. ATAC-seq peaks were identified using findPeaks.pl in HOMER with the settings: -style histone -size 75 -minDist 75 -minTagThreshold 6 -L 8 -F 8. BED files were created from Tag directories using the HOMER package. Annotation on ATAC-seq peaks or on ChIP-seq Peaks from previously published ChIP-seq data retrived from GEO (EBF1: GSE69227, PAX5:GSE38046, GATA3:GSE31235, TCF1:GSE46662, CEBPa:GSM537983) was done in HOMER using the annotatePeaks.pl command. Peakfinding on data from GEO was also performed in HOMER using the findPeaks.pl command with the option -style factor.
Genome_build: mm10
Supplementary_files_format_and_content: Processed files include .txt files with ATAC data on annotated peak positions and .txt files of RNA-seq expression matrix. All genomic coordinates are relative to mm10 mouse assembly.

Submission date

Nov 29, 2016

Last update date

May 15, 2019

Contact name

Rajesh Somasundaram

E-mail(s)

rajesh.somasundaram19@gmail.com

Phone

0046708890787

Organization name

Linkoping University