GSM9003646: D10_lumbar_1; Gallus gallus; RNA-Seq
Dissected caudal LMCs were transferred into 3ml preheated dissociation media (FACSmax™ media, Amsibio with 0,375 mg/ml Papain, Millipore Sigma) and incubated for 5 minutes in a water bath at 37°C. First and second trituration were performed with a P1000 pipette tip cut 5mm from the tip and quickly burnt with a Bunsen burner to eliminate sharp edges. The last two triturations were carried out with an uncut P1000 pipette tip. Each trituration was followed by 5 mins of incubation at 37°C. Cell suspensions were centrifugated at 300g for 7 min at 4°C. The supernatantwas discarded, cell pellets were resuspended in 1ml HBSS 5% FBS (ThermoFisher), passed through a cell strainer (Flowmi®, Millipore Sigma) into a 1,5 ml Eppendorf tube and cen-trifuged again at 300g for 7 mins at 4°C. Cell pellets were resuspended in 100μl HBSS 5% FBS and cell number and viability were assessed with an automated cell counter (Cellome-ter®, AOPI ViaStain™). Only samples above 80% viability were processed for sequencing. Chromium Next GEM Single-cell 3 Reagent Kits v3.1 (Dual Index)