Illumina HiSeq 2500 sequencing; Sox2 is present in RNA binding complexes and interacts with snRNA 7SK in mouse embryonic stem cells
Sox2 is present in RNA binding complexes and interacts with snRNA 7SK in mouse embryonic stem cells
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).
Experimental Factor: compound
dimethyl sulfoxide
Experimental Factor: immunoprecipitate
7SK