GSM5242011: E14_#3_cochlea and DRG; Mus musculus; RNA-Seq
Indrop Single cell v3 2 cochlea out of the inner ear and 6 lumbar DRGs (3 from each side) and 2 SN (1 from each side) per mouse were dissected. The dissections were done in ice-cold Ca2+-free HBSS solution (20mM HEPES in Ca2+-free HBSS). For all dissociations, enzyme mix included collagenase IV, papain and DNase I (Worthington Papain dissociation system) in HBSS solution with Ca2+ (20mM HEPES in HBSS with Ca2+ (Life Technologies) with only P14 dissociation also included trypsin. Tissues were incubated with the enzyme mix at 37C for 10-40min depending on the animal stage and triturated in between. Equal volume of ovomucoid inhibitor in EBSS was then added to stop the reaction. The cells were washed with 2% BSA in HBSS solution by spinning at 300rpm for 5-7min, resuspended with 2% BSA in HBSS. The cells were spinned through a tube with cell-strainer cap (Catalog# 352235) and loaded to FACS instrument for sorting for GFP+ glia and Neurog1cre, tdTomato+ neurons. After FACS, the cells were pelleted and resuspended in Optiprep/BSA solution (20% optiprep, 2% BSA in HBSS). An equal percentage of high GFP and low GFP glia from cochlea were combined. For both DRG+SN and cochlea samples, 2.5-5% neurons from the sorting were added to the glial cells to spike the runs as negative control. Cell mixes were loaded at 80,000 cells/ml concentration to the inDrop chip.