Illumina NovaSeq 6000 paired end sequencing; Single-cell RNAseq of Mixl1 knockout chimeric mouse embryos at embryonic day 8.5 of mouse development.
Single-cell RNAseq of Mixl1 knockout chimeric mouse embryos at embryonic day 8.5 of mouse development.
TdTomato-expressing mouse embryonic stem cells (ESC) were derived as previously described (Pijuan-Sala et al., 2019). Briefly, ESC lines were derived from E3.5 blastocysts obtained by crossing a male ROSA26tdTomato (Jax Labs – 007905) with a wildtype C57BL/6 female, expanded under the 2i+LIF conditions (Ying et al., 2008) and transiently transfected with a Cre-IRES-GFP plasmid (Wray et al., 2011) using Lipofectamine 3000 Transfection Reagent (ThermoFisher Scientific, #L3000008) according to manufacturer's instructions. A tdTomato-positive, male, karyotypically normal line, competent for chimaeras generation as assessed using morula aggregation assay, was selected for targeting Mixl1. Two guides were designed using the http://crispr.mit.edu tool  and were cloned into the pX458 plasmid (Addgene, #48138) as previously described (Ran et al., 2013). The obtained plasmids were then used to transfect the cells and single transfected clones were expanded and assessed for Cas9-induced mutations. Genomic DNA was isolated by incubating cell pellets in 0.1 mg/ml of Proteinase K (Sigma, #03115828001) in TE buffer at 50°C for 2 hours, followed by 5 min at 99°C. The sequence flanking the guide-targeted sites was amplified from the genomic DNA by polymerase chain reaction (PCR) in a Biometra T3000 Thermocycler (30 sec at 98°C ; 30 cycles of 10 sec at 98°C, 20 sec at 58°C, 20 sec at 72°C; and elongation for 7 min at 72°C) using the Phusion High-Fidelity DNA Polymerase (NEB, #M0530S) according to the manufacturer's instructions. Primers including Nextera overhangs were used, allowing library preparation with the Nextera XT Kit (Illumina, #15052163), and sequencing was performed using the Illumina MiSeq system according to manufacturer's instructions. ESC clones showing frameshift mutations in the Mixl1 coding region resulting in the functional inactivation of Mixl1 were selected for injection into C57BL/6 E3.5 blastocysts. Chimeric embryos were harvested at E8.5, dissected, and single-cell suspensions were generated from three independent pools of embryos by TrypLE Express dissociation reagent (Thermo Fisher Scientific) incubation for 7-10 minutes at 37°C under agitation. Single-cell suspensions were sorted into tdTomato+ and tdTomato- samples using a BD Influx sorter with DAPI at 1µg/ml (Sigma) as a viability stain for subsequent 10X scRNA-seq library preparation (version 3 chemistry) Performed according to manufacturer instructions (10X single-cell 3' v3 chemistry) Performed according to manufacturer instructions (10X single-cell 3' v3 chemistry)