title: 7sk_3a
organism: Mus musculus
description: Protocols: Oct4-GIP mouse embryonic stem (ES) cells (Ying et al. 2002) were cultured in either standard ES cell medium (GMEM with 10% fetal calf serum (Biosera), 0.1mM non-essential amino acids, 2mM L-Glutamine, 1mM sodium pyruvate, 0.1mM beta-mercaptoethanol and 106 units/L LIF (ESGRO, Millipore)), or in 2i/LIF medium (GMEM with 10% Knock-Out Serum Replacement (Life Technologies), 1% fetal calf serum (Biosera or Sigma), 0.1mM non-essential amino acids, 2mM L-Glutamine, sodium pyruvate, 0.1mM beta-mercaptoethanol, 1?M PD0325901 (AxonMedChem), 3?M CHIR99021 (AxonMedChem) and 106 units/L LIF (ESGRO, Millipore)). 1?g/ml puromycin was added to Oct4-GIP ES cell cultures during expansion. Cells were transfected with 1000 pmol antisense oligonucleotides (Integrated DNA Technologies) using the Mouse ES Cell Nuclefector Kit (Lonza). Total RNA was isolated using miRNeasy extraction kit (Qiagen) with in-column DNase treatment. Total RNA was depleted from ribosomal RNA with the Low Input Ribo-Zero rRNA removal system (Epicentre). rRNA-depleted samples were then fragmented with RNA fragmentation reagent (Ambion), purified using the RNeasy MinElute Kit (Qiagen) and treated with alkaline phosphatase (New England Biolabs) for 30 minutes at 37?C. 5' dephosphorylated RNA was treated with T4 PNK Kinase (New England Biolabs) for 60 minutes at 37?C. The resulting RNA (5' mono-phosphorylated and 3? OH) was purified with RNeasy MinElute (Qiagen) and ligated with RNA 3? and 5' TruSeq adapters (Illumina). Indexes 1-6 were used for PCR amplification.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2013-09-08T17:00:39Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T16:20:59Z
attributes.tag: External Id
attributes.value: SAMEA2060133
attributes.tag: INSDC center name
attributes.value: EMBL-EBI
attributes.tag: INSDC first public
attributes.value: 2013-09-08T17:00:39Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T16:20:59Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-1585:7sk_3a
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: Embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: phenotype
attributes.value: normal
attributes.tag: sample name
attributes.value: E-MTAB-1585:7sk_3a
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: 129/Ola