organism: Homo sapiens
description: Protocols: FUCCI hESCs were washed with PBS and collected by incubating with Cell Dissociation Buffer (Gibco) for 10 min at 37°C. Cells were washed with 1% PBS/BSA and crosslinked in 1% PBS/Formaldehyde, rocking for 10 min at room temperature. All the experiments were performed with single crosslinking and neutralised with Glycine (0.125M), for 5 min shaking at room temperature. Cells were washed with 1% PBS/BSA, then incubated in 1% PBS/BSA with anti-TRA-1-60-antibody mouse monoclonal IgM (1:100, Santa Cruz Biotechnology, Inc.) for 20 min on ice, together with the secondary antibody, Alexa Fluor® 647 goat anti-mouse IgM (1:1,000, Thermo Fisher) to remove background differentiation. Cells were washed with PBS, re-suspended in 3ml of PBS and sorted into the different cell cycle phases using BD FACS AriaTM III, BD Fusion or BD Influx sorters by the Cambridge NIHR BRC Cell Phenotyping Hub. Chromatin immunoprecipitation (ChIP) was performed using LowCell# ChIP kit (C0101007, Diagenode) and DNA recovered using iPure kit (C03010012, Diagenode) according to manufacturer's instructions, with the following modifications. 5μg of antibody were used per IP, and the quantity of magnetic beads adjusted accordingly, based on their binding capacity (10μl of beads bind ~3μg). Elution was made in Buffer C from the iPure kit, compatible with qPCR and next generation sequencing. For all the experiments, the same number of cells was used per IP after sorting, normalised for the less abundant cell cycle phase, usually EG1 ~1M cells. Library preparation and sequencing were performed at the Wellcome Trust Sanger Institute next-generation sequencing facility. Size selection was applied, and fragments between 100bp and 400bp (average length of 200bp) were used to prepare barcoded sequencing libraries using NEBNext Sample Prep Kit1 (NEB) following manufacturer's instructions. 10ng of input material (as a pool of the different phases) for each experiment was processed in parallel as control.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-07-24T04:03:35Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-09-18T17:50:41Z
attributes.tag: External Id
attributes.value: SAMEA5785308
attributes.tag: INSDC center name
attributes.value: MRC WT Cambridge Stem Cell Institute Department of Surgery
attributes.tag: INSDC first public
attributes.value: 2020-07-24T04:03:35Z
attributes.tag: INSDC last update
attributes.value: 2019-09-18T17:50:41Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8358:OCT4_LG1_1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: H9
attributes.tag: cell type
attributes.value: embryonic stem cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: sample name
attributes.value: E-MTAB-8358:OCT4_LG1_1
attributes.tag: scientific_name
attributes.value: Homo sapiens