Chromium single cell 3 v2

scRNA-seq experiments were performed in single replicates that were jointly processed to avoid batch effects. E11.5 limb buds of wild-type, Dup and Inv1 embryos were microdissected in 1xPBS at room temperature. A single-cell suspension was obtained by incubating the tissue for 10 min at 37 °C in 200 μl Gibco trypsin-EDTA 0.05% (Thermo Fisher Scientific, Cat. #25300054) supplemented with 20 μl 5% BSA. Trypsinization was then stopped by adding 400 μl of 5% BSA. Cells were then resuspended by pipetting, filtered using a 0.40 μm cell, washed once with 0.04% BSA, centrifuged for 5 min at 150 x g, then resuspended in 0.04% BSA. The cell concentration was determined using an automated cell counter (Bio-Rad) and cells were subjected to scRNA-seq (10x Genomics, Chromium Single Cell 3′ v2; one reaction per time point and per strain has been used) aiming for a target cell recovery of up to 10,000 sequenced cells per sequencing library (time point and strain). Single-cell libraries were generated according to the 10x Genomics instructions with the following conditions: 8 PCR cycles were run during cDNA amplification and 12 PCR cycles were run during library generation and sample indexing to increase library complexity. Libraries were sequenced with a minimum of 230 million 75 bp paired-end reads according to standard protocols.