GSM6833480: rttaControl-rep1; Mus musculus; RNA-Seq
En1-cKO and Control pups were euthanized at P2.5 and volar hindlimb skin (right and left) was rapidly dissected on ice and snap frozen on dry ice and stored at -80ºC until nuclear isolation. Single nuclear suspensions were generated from the pooled volar hindlimb skin (right and left) from 5 mice of the same genotype, for a total of 10 pooled volar skins, as previously described (https://www.protocols.io/view/snucdrop-seq-protocol-n2bvjr36plk5/v2) with some modifications. Pooled samples were dissociated in homogenization buffer using a Polytron homogenizer (Kinematica Inc.) followed by dounce homogenization (20 times with loose pestle; 10 times with tight pestle). For P2.5 volar skin, we obtained ≥ 2.1x105 nuclei/ml for each sample. Sucrose gradient centrifugation was not performed on single nuclear suspensions. Biological replicates were prepared and sequenced in two separate batches; one replicate of each genotype (En1-cKO: tetO-Cre/+; Krt5-rtTA/+; En1flox/flox; Control: En1flox/flox, Krt5-rtTA/+; En1flox/flox) was processed and sequenced in the same batch to avoid autocorrelation of condition and batch effects Single nuclei were co-encapsulated with barcoded beads and reverse transcription was performed as described here: https://www.protocols.io/view/snucdrop-seq-protocol-n2bvjr36plk5/v2 snRNA-seq (Drop-seq)