organism: Mus musculus
description: Protocols: Cells were directly lysed on the plate with RLT lysis buffer (Qiagen RNeasy Plus Mini Kit, cat. 74134). The material was transferred to microcentrifuge tubes and stored at -80°C before RNA extraction. ESC lines were grown in gelatinized dishes in Glasgow MEM (Gibco, UK) supplemented with 15% fetal bovine serum (Hyclone; ThermoFisher Scientific Scientific, UK), 1% sodium pyruvate, 1% non-essential amino acids, 0.1% β-mercaptoethanol and leukemia inhibitory factor (LIF). For RNA-seq, ESC lines were grown for 7-10 days to obtain a healthy, fast growing culture. Then 6x105 cells were seeded into wells of 6-well plates and were grown for 24h before sample collection. For each cell line, four independent cell seedings were performed to obtain replicates. Total RNA was extracted using the Qiagen RNeasy Plus Mini Kit (cat. 74134), following manufacturer's instructions. or each library, 1µg of total RNA was used with the addition of 2µl of RNA Sequins spike-in mix A/B diluted 1:25 (Garvan Institute of Medical Research, Australia). Ribosomal RNA depleted RNA-seq libraries were generated using the KAPA RNA Hyperprep kit with RiboErase (Roche, cat. KK8560), following manufacturer's instructions. Each RNA-seq library was uniquely indexed using KAPA Dual-Indexed adapters (Roche, cat. KK8722). Libraries were quantified using a Qubit Fluorometer (Qubit dsDNA High Sensitivity kit) and fragment size was evaluated using an Agilent 2100 Bioanalyser (Agilent High Sensitivity DNA kit).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-05-01T04:02:51Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-02-11T14:10:00Z
attributes.tag: External Id
attributes.value: SAMEA5331855
attributes.tag: INSDC center name
attributes.value: Bioinformatics Researcher
attributes.tag: INSDC first public
attributes.value: 2019-05-01T04:02:51Z
attributes.tag: INSDC last update
attributes.value: 2019-02-11T14:10:00Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7655:ZFC4mut_4
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: ES cell line
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: genotype
attributes.value: ZFC4mut
attributes.tag: sample name
attributes.value: E-MTAB-7655:ZFC4mut_4
attributes.tag: scientific_name
attributes.value: Mus musculus