GSM9379892: 1DPA, mouse limb cultured in 96 well plate - replicate 1; Mus musculus; ATAC-seq
For bulk ATAC sequencing, 4 mouse limbs each from ALI and 96-well cultures were collected at 1 dpa in 1.5 ml Protein LoBind Tubes (Eppendorf 0030108442) and washed in 1 ml of PBS 1x at room temperature. Limbs were dissociated by incubation in 200 µl of 0.25% Trypsin-EDTA (Gibco 25200-072) for 6 min at 37 °C, followed by gentle pipetting. The enzymatic reaction was stopped by the addition of 400 µl DMEM base, and diluted with 800 µl of PBS 1x. The cells were pelleted by centrifugation for 5 min at 400 rcf. Nuclei extraction was performed using the Diagenode ATAC-seq kit (Diagenode C01080002) Sequencing libraries were generated using the Diagenode ATAC-seq kit (Diagenode C01080002) and i7 and i5 index primers described previously (Zenk et al., 2023). Quality control of sequencing libraries was performed using the Qubit dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit (Agilent 5067-4626) on the Agilent 2100 Bioanalyzer system. A 15 nM equimolar pool of samples was submitted to GENEWIZ for sequencing. The sequencing libraries, bearing dual combinatorial indices and with a 5% PhiX spike-in, were loaded on a 25B flow cell on the Illumina NovaSeq X Plus instrument according to the manufacturer's instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration v1.5. Image analysis and base calling were conducted by the NovaSeq Control Software v1.8.1 on the NovaSeq instrument. Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification.