title: NextSeq 500 sequencing; RNA-Seq of mouse embryonic stem cells derived from genetically diverse inbred mouse strains with varying alleles at Lifr gene
design: RNA-Seq of mouse embryonic stem cells derived from genetically diverse inbred mouse strains with varying alleles at Lifr gene
library_construction_protocol: 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.