GSM6499476: e16.5, mutant, rep1; Mus musculus; RNA-Seq
The thalamus was manually dissected from control (ShhΔSBE1;ΔSBE5/+) and mutant (ShhΔSBE1;ΔSBE5/ΔSBE1;ΔSBE5) brains at four embryonic stages (E12.5, E14.5, E16.5 and E18.5) in ice-cold PBS. The caudal and rostral boundaries of dissection coincided with the cephalic flexure and the mammillary body, respectively. Each thalamus was cut into small pieces and dissociated into a single cell suspension using the Papain Dissociation System (Worthington, LK003153) according to the manufacturer's protocol. Samples were dissociated in Papain-EBSS solution for 40 minutes at 37ºC. Papain was inactivated with ovomucoid protease inhibitor, and the digested tissue was resuspended in PBS (calcium and magnesium free) containing 0.04% weight/volume BSA (400 µg/ml). Cell suspensions were stained with Trypan Blue to determine the ratio of viable to damaged cells and counted using a haemocytometer (Thermo Fisher Scientific). Single cell suspensions containing more than 90% viable cells were fixed in methanol for 1 week. Samples were rehydrated at a concentration of 700-1200 cells/l in ice-cold PBS containing 0.04% weight/volume BSA (400 µg/ml) immediately prior to the generation of single-cell RNA sequencing libraries. Single-cell RNA-seq libraries were generated using the 10X Genomics platform (Chromium Single Cell 3' library and Gel Bead Kit v3, PN-1000075) and sequenced on a NovaSeq 6000 system (Illumina) at the Center for Applied Genomics (Children's Hospital of Philadelphia). Three independent libraries were generated for each time point and genotype (n=24 libraries).