GSM5627616: AB5786; Mus musculus; RNA-Seq
Forelimb tissues were disassociated using enzymatic digestion. E10.5-E11.5 forelimbs were digested with pre-heated 0.25% trypsin in DMEM medium (ThermoFisher) and incubated for 10 min at 37°C, gently pipetting every 3 min. E12.5-E14.5 forelimbs were digested with 1.5 mg/ml  collagenase type V (Sigma-Aldrich) in DMEM at 37°C for 10-15 min, gently pipetting every 5 min until the tissue completely dissolved. The digestion reaction was stopped by addition of DMEM supplemented with 10% FBS and 1% Pen-Strep. Cell suspensions were filtered through a 40-µm nylon mesh and collected by centrifugation at 1000 rpm for 7 min at 4°C. Supernatant was removed and cells were resuspended in 500 µl ice-cold MACS buffer (with 0.5% BSA and 2 mM EDTA in PBS) and used immediately for fluorescence-activated cell sorting (FACS). Samples were flow sorted using AriaIII (BD Biosciences, BD Diva Software) cell sorter. 3' end mRNA libraries were prepared for sequencing using MARS-seq (Jaitin et al, Science 2014)