title: Single-cell RNA-seq of axolotl regenerating and control thymus
design: Single-cell RNA-seq of axolotl regenerating and control thymus
library_construction_protocol: 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.   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). 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.
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-16013:L40849_Axo_5dpt_p