GSM9378077: P3 digit, SAR131675 diet; Mus musculus; RNA-Seq
Digits from ten mice were pooled for each sample. A scalpel blade was used to sever hindlimb digits 2-4 at the proximal end of the 2nd phalangeal element. Each digit was divided down the midline along its length to maximize the exposure of internal tissue surface area to media. A single cell suspension was generated following these techniques: Briefly, digit tissue was subjected to enzymatic digestion using Collagenase I at 3 mg/mL (Thermo Fisher Scientific, 17100-017), Collagenase II at 2 mg/mL (Thermo Fisher Scientific, 17101-015), and Dispase at 3mg/mL (MilliporeSigma, D4693) per 1mL of DMEM (Thermo Fisher Scientific, 11960044) for 45 minutes at 37°C under constant agitation at 150 rpm. Digestions were then quenched with DMEM containing 10% FBS (Gibco, 10437028), filtered through 40um mesh and pelleted via centrifugation for 5min at 400xG at 4⁰C.Supernatant was removed and red blood cells were lysed by resuspending the pellets with 2ml of ACK Lysis Buffer (Gibco A10492-01) and incubating for 2 minutes at room temp. Cell viability was assessed with Trypan blue exclusion on a Countess III (Thermo Fisher Scientific) automated counter and only samples with >75% viability were processed for further sequencing. Single Cell 3' v3