title: LIFKI002N-1x-3_GT19
organism: Mus musculus
description: Protocols: Euploid (>70%), germline competent, male mESCs were derived from blastocysts of 129S1/SvImJ and NOD/ShiLtJ mice. CRISPR/Cas9 donors and sgRNAs were designed to engineer reciprocal reference and alternate alleles at SNP rs50454566. A 128bp oligo donor was selected (sequences provided below). A 20bp guide (GGGAGACAGAGCTTTCGACA - reference allele or GGAGACACAGCTTTCGACA - alternate allele) was cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene #62988). 2×10^6 cells seeded on a 60mm dish were co-transfected with equimolar amounts (2.5ug each) of donor and guide vector using Lipofectamine 3000 (Invitrogen #L3000) reagent. After 48 hours, cells underwent puromycin selection and after one week colonies were picked and expanded. Clones were PCR amplified using 5'-AGGCCCAGAGTGATTGACTT-3' and 5'-GGACACAGCACCCAGATTTC-3' and capillary sequencing of the resulting PCR products was used to identify correct, homozygous targeting. Donors oligos were as follows: 129S1 SNP, 5'-GAAACACAATAGCAGGCT[T]CAGTTAAGTGACTTTGACCTTGTCGAAAGCTGTGTCTCCCTTTTGAGATGTTAAAAGTTTCAGGAAAAGAAGCACACTAAAAGAAATCTGGGTGCTGTGTCCACCCCCA-3'. NOD SNP: 5'-GAAACACAATAGCAGGCT[A]CAGTTAAGTGACTTTGACCTTGTCGAAAGCTCTGTCTCCCTTTTGAGATGTTAAAAGTTTCAGGAAAAGAAGCACACTAAAAGAAATCTGGGTGCTGTGTCCACCCCCA-3'. For collection of samples for RNA-seq data generation, 1.5×10^4 cells were plated onto gelatinized 35mm tissue culture treated dishes in 1i/LIF (CHIR99021). Cells remained unfed until harvest, six days later. Cell lines were untreated. RNA was harvested using the RNeasy (Qiagen) RNA extraction kit Poly(A) RNA-Seq libraries were constructed using the KAPA mRNA HyperPrep Kit (KAPA Biosystems). Libraries were checked for quality and quantitated with the Agilent Bioanalyzer, and samples that failed QC were repeated starting from the cryovial stage.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-08-13T04:07:11Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-01-17T16:19:23Z
attributes.tag: External Id
attributes.value: SAMEA6489534
attributes.tag: INSDC center name
attributes.value: Jackson Laboratory
attributes.tag: INSDC first public
attributes.value: 2020-08-13T04:07:11Z
attributes.tag: INSDC last update
attributes.value: 2020-01-17T16:19:23Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8695:LIFKI002N-1x-3_GT19
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: developmental stage
attributes.value: blastocyst
attributes.tag: genetic modification
attributes.value: allele swap at rs50454566 from 129S1/SvImJ
attributes.tag: genotype
attributes.value: 129S1 allele at SNP rs50454566
attributes.tag: sample name
attributes.value: E-MTAB-8695:LIFKI002N-1x-3_GT19
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: NOD/ShiLtJ