title: Sample 3
organism: Mus musculus
description: Protocols: ATAC-seq was performed on ESCs and at each day of the differentiation following methods previously described by Buenrostro et al, 2013. Briefly, adherent cells were treated with StemPro Accutase to obtain a single cell suspension. Cells were counted and resuspended to obtain 50,000 cells per sample in ice-cold PBS. Mouse HM1 (wild type), T/Bra-/- and Cdx1,2,4-/- mutant ESC lines ( http://dx.doi.org/10.1016/j.devcel.2017.04.002 Gouti et al, 2017) and inducible Cdx2 ESCs (5ECER5G20, Niwa et al 2005, Cell DOI: 10.1016/j.cell.2005.08.040), were propagated on mitotically inactivated mouse embryonic fibroblasts (feeders) in DMEM knockout medium supplemented with 1000U/ml LIF, 10% cell-culture validated fetal bovine serum, penicillin/streptomycin, 2mM L-glutamine. To obtain neural progenitors with anterior, hindbrain or spinal cord neural identity, ESCs were differentiated as previously described (https://doi.org/10.1371/journal.pbio.1001937 Gouti et al, 2014). Briefly, ESCs were dissociated with 0.05% trypsin, and plated on gelatin-coated plates for two sequential 20 minute periods in ESC medium to separate them from their feeders. To start the differentiation, cells remaining in the supernatant were pelleted by centrifugation, washed in PBS, and pelleted again. Cells were counted and resuspended in N2B27 medium containing 10ng/ml bFGF to a concentration of 106 cells per ml, and 50,000 cells per 35mm CELLBIND dish were plated. To generate anterior neural progenitors, the cells were grown up to day 3 in N2B27 + 10ng/ml bFGF, followed by N2B27 + 500nM smoothened agonist (SAG; Calbiochem) from day 3-5. To generate hindbrain neural progenitors, cells were cultured under the same conditions as the anterior, but were additionally exposed to 100nM retinoic acid (RA; Sigma) from day 3-5. To generate spinal cord neural progenitors, cells were cultured with N2B27 + 10ng/ml bFGF until day 2, N2B27 + 10ng/ml bFGF + 5 micro M CHIR99021 until day 3, and N2B27 + 100nM RA + 500nM SAG until day 5. For the inducible CDX2 experiments, induction of CDX2 was performed using 5ECER4G20 ESCs (Niwa et al., 2005) by adding 1ug/ml of 4-hydroxy tamoxifen (Sigma) (Niwa et al., 2005) between D2-3 (in the presence of 10ng/ml bFGF in N2B27) or D4-5 (in the presence of 10ng/ml bFGF + 100nM RA + 500nM SHH in N2B27). For all differentiations, media changes were made every 24 hours from day 2. Cells were pelleted and resuspended in lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL). Following a 10 min centrifugation at 4 degrees C, nucleic extracts were resupsended in transposition buffer (Illumina) for 30 minutes at 37 degrees C and purified using a Qiagen MinElute PCR Purification kit following manufacturer's instructions. Transposed DNA was eluted in a 10 micro l volume. Transposed DNA was amplified by PCR with Nextera primers to generate single-indexed libraries. A maximum of 12 cycles of PCR (determined using optimisation experiments) was used to prevent saturation biases based on optimisation of qPCR cycles as previously described in Beunrostro et al 2015. Library quality control was carried out using the Bioanalyzer High-Sensitivity DNA analysis kit as per manufacturer's instructions.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-08-28T17:03:19Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-07-02T17:48:24Z
attributes.tag: External Id
attributes.value: SAMEA4774556
attributes.tag: INSDC center name
attributes.value: The Francis Crick Institute
attributes.tag: INSDC first public
attributes.value: 2018-08-28T17:03:19Z
attributes.tag: INSDC last update
attributes.value: 2018-07-02T17:48:24Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6337_3:Sample 3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HM1 ESC
attributes.tag: cell type
attributes.value: mouse embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: HPRT negative
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: sample name
attributes.value: E-MTAB-6337_3:Sample 3
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: 129x129