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To generate 1.8-Nanog-mCherry and 1.8-Esrrb-mCherry reporter lines, 1 × 106 1.8 mESCs were transfected with 4 μg of the pUC19-Nanog-mCherry-puro or pUC19-Esrrb-mCherry-puro plasmid (Additional file 9: Table S8) and 1.5 μg of each of the sgRNAs plasmids (PX330-Nanog-sgRNA1/2 and PX330-Esrrb-sgRNA1/2) using 16.5 μl of Lipofectamine 3000 and 22 μl of P3000 (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Cells were selected with puromycin (1 ng/μl, Sigma) for 3 days, starting at day 2 after transfection. The puromycin selection cassette was subsequently excised by transient transfection of a CRE recombinase expression plasmid pCAG-Cre (Addgene 13,775, [94]). Individual clones were expanded and tested for loss of puromycin resistance. mCherry fluorescence was measured via flow cytometry and clones were subsequently genotyped by PCR (Additional file 1: Figure S2B). All PCRs were carried out by using the Hotstart Taq Polymerase (Qiagen), a Tm of 56 °C and 30 cycles (Primer sequences are listed in Additional file 9: Table S8).

In order to generate Klhl13 mutant mESCs, 4 guide RNAs were designed to target a 4.5-kb region around the Klhl13 promoter (2 guide RNAs on each side) with the Alt-R® CRISPR-Cas9 System (IDT), which contains all necessary reagents for the delivery of Cas9-gRNA ribonucleoprotein complexes (RNP) into target cells. Briefly, crRNAs and tracrRNA (gRNA sequences in Additional file 9: Table S8) were mixed in equimolar concentrations and the 4 crRNAs and tracrRNA duplexes were subsequently pooled together. 2.1 μl PBS, 1.2 μl of the tra + cr duplex (100 μM stock), 1.7 μl Cas9 (61 μM stock), and 1 μl electroporation enhancer were pipetted together and incubated for 20 min. In total, 105 cells were nucleofected with the mixture using the CP106 program of the Amaxa 4D-Nucleofector (Lonza) and plated on gelatin-coated 48-well plates. After 48 h, cells were seeded at a density of 10 cells/cm2 into 10-cm plates. Individual clones were picked, expanded, and genotyped for the presence of the promoter deletion. The genotyping strategy is shown in Additional file 1: Figure S4B. For the amplification of the wildtype band, the HotStart Taq Polymerase (Qiagen) was used with an annealing temperature of 51 °C and 35 cycles. For the deletion, the Phusion HiFi Polymerase (NEB) was used with an annealing temperature of 63 °C and 35 cycles (Primer sequences are listed in Additional file 9: Table S8).

For the generation of Dusp9 mutant mESCs, 2 × 106 WT and K13-HET (Clone 1) cells were nucleofected with 5 μg of the PX458-Dusp9_sgRNA1 plasmid (Additional file 9: Table S8) and subsequently plated on gelatin-coated 6-cm plates. The next day, high GFP+ cells were single-cell sorted into a 96-well plate and expanded. Clones were screened for homozygous or heterozygous frameshift deletions via Sanger sequencing and immunoblotting. Heterozygous deletion of several selected clones was further confirmed via NGS. Briefly, a region surrounding the Dusp9 deletion was amplified using the Phusion HiFi Polymerase (NEB) with a total of 30 cycles and an annealing temperature of 65 °C (Primer sequences in Additional file 9: Table S8, OG197/OG198). A second PCR using again the Phusion HiFi Polymerase (NEB) with a total of 14 cycles and an annealing temperature of 65 °C was performed in order to attach the Illumina adaptors and barcodes (Additional file 9: Table S8, OG202/OG210). A dual barcoding strategy was employed, where Illumina barcodes were included in the reverse and custom sample barcodes in the forward primers. Samples containing the same Illumina barcode but different custom sample barcodes were pooled in an equimolar fashion and sequenced on the Illumina Miseq platform PE150. Samples were aligned using Bowtie2 [95] and an index containing sample barcodes and possible deletion sequences based on previously generated Sanger sequencing data, gaining approximately 4000 reads per sample.

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