title: 18858_6#74
organism: Mus musculus
description: Protocols: Wildtype E14 mESC were cultured under standard culture conditions in Knockout DMEM containing 15% Fetal Calf Serum, 1x Penicillin-Streptomycin-Glutamine, 1x NEAA 2-mercaptoethanol and 1000U Leukemia Inhibitory Factor. Trypisnised single mESC suspension is passed through 30micron filter to remove and clumps. A 96-well plate containing lysis buffer (0.2% Triton, 1:20 RNAse inbhibitor, 10mM Oligo-dT30VN, 10mM dNTPs) was prepared. Spike-ins (ERCCs and SIRVs; final 1:1000,000 dilution) are subjected to different freeze-thaw cycles (seven conditions) and 1ul of spike-in mix per condition was added to each well column-wise. Single mES cells were FACS sorted (BD Influx; BD Biosciences) to each well (along with bulk controls). Subsequent steps (Lysis, RT, PCR amplification and DNA purification) was performed as described in Smart-Seq2 publication (Picelli et al Nature Protocol 2014). The single cell cDNA across the 96-well plate was quantified, diluted and libraries were made using Nextera XT DNA sample preparation kit (Illumina) utilising 96 dual barcoded indices. Library pooling was performed post AMPure XP beads (Agencourt Biosciences) clean up and sequenced on HiSeq2500 (v4) using paired end sequencing (75 or 125bp) at the Wellcome Trust Sequencing facility following Wellcome Trust Sequencing guidelines.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2017-03-15T17:01:09Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2017-03-14T21:38:39Z
attributes.tag: External Id
attributes.value: SAMEA103911040
attributes.tag: INSDC center name
attributes.value: Wellcome Trust Sanger Institute
attributes.tag: INSDC first public
attributes.value: 2017-03-15T17:01:09Z
attributes.tag: INSDC last update
attributes.value: 2017-03-14T21:38:39Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5481:18858_6#74
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: ES-E14
attributes.tag: cell type
attributes.value: mouse embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: growth condition
attributes.value: Serum + LIF condition
attributes.tag: sample name
attributes.value: E-MTAB-5481:18858_6#74
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: 129/Ola