biostudies-arrayexpressMaximina YunAmbystoma mexicanumhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16013For 5 dpt, 10 dpt, 21 dpt and 35 dpt samples for scRNA-seq the whole region of thymectomy was collected to include the thymic nodules and surrounding tissues using dissection forceps and scissors. For the control and regenerated thymic nodules at 60 dpt only the thymic nodules themselves were collected excluding any additional surrounding tissue. Six thymic nodules or six thymic area samples were pooled per conditionbiostudies-arrayexpressSequencing - Basecalls were done with Illumina bcl2fastq version 2.19.1 (files with library id L53237 or lower) or Illumina bcl2fastq version 2.20.0 (files with library id higher than L53237)Sample Collection - Thymectomised or control animals were collected and the samples were dissected to remove either the thymus nodules individually or the whole thymus region including other tissues. Six thymic nodules or six thymic area samples were pooled per condition, washed in A-PBS (0.8X PBS), and incubated in 1 mL of 1x LiberaseTM research grade (Sigma) for 45 minutes at room temperature with rotation and additional manual dissociation using forceps. Cells in solution were mixed by gentle pipetting and filtered through a 70 µM Filcon (BD™ Medimachine) and collected into a falcon tube. DAPI (Thermo-Fisher) was added at 1:1000 and the solution was kept on ice until FACS sorting. The following steps were performed by the FACS facility and Dresden Concept Genome Centre, core facilities of the CMCB Technology Platform at the Technische Universität Dresden. Up to 30,000 cells were sorted for living cells into a 1.5 ml Eppendorf tube containing 5 µl A-PBS with 0.04% BSA with FACS Aria II. The cells were concentrated by centrifugation (300 rcf, 5 min, 4 °C) and the volume reduced to ~42 µl. Cells were carefully resuspended and visually inspected under a light microscope to determine cell concentration and quality.Library Construction - The 10X Genomics single cell RNA-seq library preparation - involving fragmentation, dA-Tailing, adapter ligation and 10-12 cycles indexing PCR – was performed based on the manufacturer’s protocol. After quantification, the libraries were sequenced on an Illumina Novaseq6000 in 2x 100 bp paired-end mode, generating ~150-250 million fragment pairs.Nucleic Acid Extraction - The concentrations of the single-cell suspensions were adjusted to 410 cells per microliter or used directly when lower concentrated, before they were carefully mixed with the reverse transcription mix and loaded on the 10X Genomics Chromium system(1) in a Chromium Single Cell G Chip targeting up to 10,000 cells per reaction. Following the guidelines of the 10x Genomics Chromium Single Cell Kit v3.1 user manual, the droplets were directly subjected to reverse transcription, the emulsion was broken and cDNA was purified using Dynabeads MyOne Silane (provided by 10X Genomics).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The 10x sequencing data was processed with the ‘count’ command of the Cellranger software (v4.0.0) provided by 10x Genomics (https://www.10xgenomics.com/). The Cellranger reference was built with the ‘mkref’ command of the Cellranger software using the axolotl genes-only reference with improved gene annotations. Sequence and annotation of the GFP construct were included as well. Sequence and annotation (without GFP) is included as supplementary file.Sequence Alignment - Axolotl reference sequence and gene annotations (both version AmexG_v3.0.0) were downloaded from the axolotl-omics website (https://www.axolotl-omics.org/assemblies). The mitochondrial genome sequence and its gene annotations were downloaded from GenBank (accession AY659991.1) Due to the size of the genome, only genic regions were used for analysis. Gene coordinates were extended by 50kb to both sides and merged when overlapping. Sequence and gene annotation were then extracted for each genic region to build a genes-only reference. The gene annotation still lacked information about UTRs and transcript isoforms crucial for the analysis of 10x data. Therefore, gene annotations were improved as follows: Publicly-available RNA-seq datasets were downloaded from the NCBI SRA (accessions: SRR5341564-SRR5341566, SRR5341568, SRR5341569, SRR5341572-SRR5341577, SRR5341579-SRR5341584). Each dataset was mapped with STAR (v2.7.6a) to the genes-only reference to collect splice-site information which was then utilized in a second pass to improve the mappings. Transcript models were assembled per dataset using StringTie (v1.3.5) and then merged. Publicly-available de-novo axolotl transcriptome assemblies(PMID:28099853, PMID:29364872) were downloaded from the Broad institute (https://portals.broadinstitute.org/axolotlomics/) and the NCBI TSA (accession:GFZP00000000.1), respectively. Transcript contigs were mapped to the genes-only reference with BLAT (UCSC tools v416) requiring that a transcript contig aligned at least 90% of its length with a sequence identity of 90%. StringTie transcript models and BLAT-mapped transcript contigs were assembled using the PASA (Program to Assemble Spliced Alignments, v2.3.3) pipeline. The resulting PASA alignment assemblies were used for two rounds of annotation update with PASA to add UTRs and transcript isoforms to the annotation of the genes-only reference.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsAmbystoma mexicanumMaximina YunfalseSingle-cell RNA-seq of axolotl regenerating and control thymusFor 5 dpt, 10 dpt, 21 dpt and 35 dpt samples for scRNA-seq the whole region of thymectomy was collected to include the thymic nodules and surrounding tissues using dissection forceps and scissors. For the control and regenerated thymic nodules at 60 dpt only the thymic nodules themselves were collected excluding any additional surrounding tissue. Six thymic nodules or six thymic area samples were pooled per condition2025-11-12T00:00:00Z2026-05-27T14:56:26.379Z2025-11-12T09:57:38.78ZE-MTAB-16013ERP183895EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184