Illumina HiSeq 4000 paired end sequencing; Single-cell RNAseq of Brachyury knockout chimeric mouse embryos at embryonic days 8.5 and 7.5 of mouse development.
Single-cell RNAseq of Brachyury knockout chimeric mouse embryos at embryonic days 8.5 and 7.5 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 T. Two guides were designed using the http://crispr.mit.edu tool (guide 1: TGACGGCTGACAACCACCGC; guide 2: GCCCCAAAATTGGGCGAGTC) 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 (F- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCCCGGTGCTGAAGGTAAAT; R- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCTGCTTAACCCTCATCAGC), allowing library preparation with the Nextera XT Kit (Illumina, #15052163), and sequencing was performed using the Illumina MiSeq system according to manufacturer's instructions. Two ESC clones showing frameshift mutations in exon 2 resulting in the functional inactivation of T were selected for injection into C57BL/6 E3.5 blastocysts. A total of 17 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 2 chemistry) Performed according to manufacturer instructions (10X single-cell v2 chemistry) Performed according to manufacturer instructions (10X single-cell v2 chemistry)
Experimental Factor: developmental stage
embryonic day 7.5
Experimental Factor: genotype
T-/-