This dataset contains the custom genomes generated to analyse sequencing data in the manuscript titled "Temporal constraints on enhancer usage shape the regulation of limb gene transcription" by Rouco et al. 2024_2025. For more information see Materials and Methods section. The main customized genome and index used across the manuscript were generated based on the GRCm39/mm39 (Cunningham et al., 2022) incorporating the dmCherry-P2A-CRE and floxed-SV40pASTOP-EYFP cassettes as artificial chromosomes, therefore we termed it as GRCmm39/ mm39_dsmCherry_P2A_CRE_EYFP genome: mm39_dsmCherry_P2A_CRE_EYFP.fa mm39_dsmCherry_P2A_CRE_EYFP.fa.fai These files were used to build a custom reference for different downstream analyses using the following pipelines:CellRanger: cellranger_mm39_dsmCherry_P2A_CRE_EYFP.sh Bowtie2: bowtie2_mm39_dsmCherry_P2A_CRE_EYFP.sh Star: star_mm39_dsmCherry_P2A_CRE_EYFP.sh For annotation, GTF files sourced from ENSEMBL GRCm39 release 104 (Cunningham et al., 2022) were used, with a filtering process applied to exclude read-through/overlapping transcripts. Only transcripts annotated as 'protein-coding' for their respective genes were retained, while those flagged as 'retained_intron', 'nonsense-mediated decay', etc., were discarded. This filtration aimed to retain only unambiguous exons, mitigating potential quantitative biases during data analysis conducted using STAR/Cufflinks (Amandio et al., 2016). mergeOverlapGenesOfFilteredTranscriptsOfMus_musculus.GRCm39.104_ExonsOnly_UCSC_dsmCherry_P2A_CRE_EYFP.gtf The second customized genome and index used in this manuscript, for precise genotype characterization, was generated based on the GRCm39/mm39 (Cunningham et al., 2022) incorporating the dmCherry-P2A-CRE cassette at the Shox2 locus, therefore we termed it as GRCmm39/ mm39_Shox2_dsmCherry_P2A_CRE_bGHpA_KI genome: mm39_Shox2_dsmCherry_P2A_CRE_bGHpA_KI.fa mm39_Shox2_dsmCherry_P2A_CRE_bGHpA_KI.fa.fai These files were used to build a custom reference for different downstream analyses using the following pipeline: bowtie2_mm39_Shox2_dsmCherry_P2A_CRE_bGHpA_KI.sh The following GTF with the shifted coordinates accounting for the insertion of the cassette was also created: shifted_completed_mm39_Shox2_dsmCherry_P2A_CRE_KI.gtf Files with shifted coordinates following the pipeline described in: https://github.com/lldelisle/howCanYou/tree/main/NGS/mutant_genome#shift-annotations-to-work-on-your-mutant-genome