title: GSM2410028: BM-LSK-cells-undifferentiated [ATAC-seq-LSK_2]; Mus musculus; ATAC-seq
library_construction_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.
attributes.tag: GEO Accession
attributes.value: GSM2410028