title: Sample 4
organism: Mus musculus
description: Protocols: 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 all differentiations, media changes were made every 24 hours from day 2. Samples were collected as previously described (Kutejova et al., 2016). 10-30 million cells from day 5 hindbrain or day 5 spinal cord neural progenitors were collected from plates using scrapers and crosslinked in 1% formaldehyde for 20 min at 4 degrees. Chromatin was sonicated using a Diagenode Bioruptor (using a cycle of 30sec on, 30 sec off) until fragments were between 200-400bp. Samples were decrosslinked and purified using the Qiagen MinElute kit following manufacturer's instructions. Chromatin immunoprecipitation was performed as previously described in Kutejova et al., 2016. 3ug of Sox2 antibody (Santa cruz antibody SC-17320X) was incubated together with cell lysate overnight at 4°C on a rotating wheel. Immunoprecipitation of chromatin fragments were captured using Protein G-coupled Dynabeads (Life Technologies). Samples were decrosslinked and purified using the Qiagen MinElute kit as per manufacturer's instructions. To generate libraries, approximately 10ng ChIP DNA and 10ng input DNA was used for each condition. Libraries were generated by by blunt-end repair, dA-tailing, and ligation of Illumina adaptors using the KAPA Hyper Prep Kit following manufacturer's instructions. Libraries were size-selected to obtain fragments between 250-400bps using an E-Gel EX Agarose Gel. 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: 2018-08-28
attributes.tag: ENA last update
attributes.value: 2018-01-15
attributes.tag: External Id
attributes.value: SAMEA104469055
attributes.tag: INSDC center alias
attributes.value: The Francis Crick Institute
attributes.tag: INSDC center name
attributes.value: The Francis Crick Institute
attributes.tag: INSDC first public
attributes.value: 2018-08-28T17:03:14Z
attributes.tag: INSDC last update
attributes.value: 2018-01-15T13:26:26Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6348:Sample 4
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HM1 ESC
attributes.tag: cell type
attributes.value: mouse neural progenitor cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: progenitor cell type
attributes.value: mouse embryonic stem cell
attributes.tag: protocol
attributes.value: hindbrain neural progenitor differentiation
attributes.tag: sample name
attributes.value: E-MTAB-6348:Sample 4
attributes.tag: strain
attributes.value: 129x129