title: Doxy plus 7SK INPUT
organism: Mus musculus
description: Protocols: 2TS22C mES cells, where Sox2 can be deleted upon doxycycline treatment, were kindly provided by Dr. Hitoshi Niwa at the RIKEN Center for Developmental Biology, Kobe, Japan. All mES cell lines were grown on 0.1% gelatin coated plates and maintained in ES media consisting of Glasgow Minimum Essential Medium (GMEM) supplemented with 10% fetal calf serum for ESCs (Biosera, Boussen, France), 0.1 mmol/L non-essential amino acids, 2 mmol/l L-Glutamine, 1 mmol/l sodium pyruvate, 0.1 mmol/l β-mercaptoethanol, 1x penicillin/streptomycin and 106 units/L LIF (ESGRO, MilliporeCorp., Billerica, MA, USA). Mouse 2TS22C cells were cultured as above and either treated with Dimethyl sulfoxide (DMSO) or Doxycycline (1 ug/ml) for 24 h before cross-linking with glutaraldehyde. 20 million cells were used per ChIRP. Six probes covering the whole length of 7SK were used and depending on their positions along the RNA were divided into odd and even probe pools. A single probe against LacZ mRNA was used as a negative control. Isolated RNA from a small aliquot of post-ChIRP beads was used in qRT-PCR to quantify 7SK enrichment. Standard Illumina protocol DNA from ChIRP was treated with RNases A and H followed by Proteinase K. DNA was cleaned up with Phenol:Chloroform:Isoamylaclcohol extraction and the resulting aqueous phase was precipitated with 2.5 volumes of ethanol, 1/10th volume Sodium acetate (pH 5.2) and glycoblue. The pellet was washed with 70% ethanol and re-suspended in 30ul elution buffer. Isolated DNA following ChIRP was used to make sequencing library with ThruPLEX DNA-seq 12S kit (R400428, Rubicon Genomics).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-02-11T17:04:30Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-01-04T14:55:15Z
attributes.tag: External Id
attributes.value: SAMEA5205640
attributes.tag: INSDC center name
attributes.value: University of Portsmouth
attributes.tag: INSDC first public
attributes.value: 2019-02-11T17:04:30Z
attributes.tag: INSDC last update
attributes.value: 2019-01-04T14:55:15Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7570:Doxy plus 7SK INPUT
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: 2TS22C
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: Sox2Zeo
attributes.tag: phenotype
attributes.value: Sox2 deficient
attributes.tag: sample name
attributes.value: E-MTAB-7570:Doxy plus 7SK INPUT
attributes.tag: scientific_name
attributes.value: Mus musculus