title: SIGAG9
organism: Mus musculus
description: Protocols: Cell line generation Individual mouse Embryonic Stem Cell lines were derived from different blastocysts of well-defined inbred strains and genetically diverse outbred mice (Diversity Outbred) derived from the Collaborative Cross (“The Collaborative Cross, a community resource for the genetic analysis of complex traits,” 2004). Cell line maintenance and differentiation mESC lines were thawed on CF1 irradiated mouse embryonic fibroblasts in KSR medium supplemented with 10ng/mL LIF. Cells were transferred and grown in 2iL condition (N2B27 supplemented with 10ng/mL LIF, 3μM CHIR99021 and 1μM PD0325901), they were passaged every two days using TrypLE. For differentiation towards EpiSCL cells the supplements were removed for the indicated number of days. Conversion was performed following the 10X protocol (https://www.10xgenomics.com/) Chromium 10X platform. Library was constructed according to User Guide for Chromium Single Cell 3ʹ Reagent Kits v3 - CG000183 Rev A (https://assets.ctfassets.net/an68im79xiti/4tjk4KvXzTWgTs8f3tvUjq/2259891d68c53693e753e1b45e42de2d/CG000183_ChromiumSingleCell3__v3_UG_Rev_C.pdf [assets.ctfassets.net]) cDNA was always run on Tapestation 4200 with High Sensitivity D5000 assay while final libraries with High Sensitivity D1000 assay - however in few occasions Bioanalyser 2100 with High Sensitivity Kit was used for both cDNA and final libraries. After that QC final libraries were qPCR using QuantStudio 6 and Kapa Biosystems ROX LOW QPCR MASTER MIX. Basing on that quantification libraries were normalised to 15nM and pooled into SLX tubes.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-12-15T17:12:00Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-03-04T13:50:37Z
attributes.tag: External Id
attributes.value: SAMEA6602900
attributes.tag: INSDC center name
attributes.value: Wellcome Sanger Institute
attributes.tag: INSDC first public
attributes.value: 2020-12-15T17:12:00Z
attributes.tag: INSDC last update
attributes.value: 2020-03-04T13:50:37Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8844:SIGAG9
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: epiblast stem cell like cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: N2B27
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: progenitor cell type
attributes.value: embryonic stem cell
attributes.tag: sample name
attributes.value: E-MTAB-8844:SIGAG9
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: mixed CAST/EiJ, C57BL/6J, C3H/HeJ, PWD/PhJ