GSM9040538: E11.5 lambdoidal junctions epithelia replicate 1; Mus musculus; RNA-Seq
Embryonic litters were collected and staged based on somite count and limb morphology on designated days of development and then dissected in ice-cold PBS. At E9.5, embryonic faces were isolated from the heads by dissecting the frontonasal and maxillary prominences, carefully avoiding the forebrain and developing eye placode. By E10.5 and E11.5, entire midfaces were dissected to include the MNP, LNP, and MxP, while avoiding eye and brain tissues. The lambdoidal junction was specifically isolated using surgical scalpels to dissect the upper halves of the LNP and MNP at the horizontal midline of the nasal pits, and the lower half of the MxP at the horizontal midpoint, ensuring the lambdoidal junction remained intact without separating the individual prominences. For wild-type (Swiss Webster) scRNAseq, samples were pooled for dissociation into single cell suspensions. A fresh cocktail of 1X Liberase TL (Roche #05401020001) and 1X DNAseI Grade II (Roche #10104159001) was prepared. Tissue samples were then incubated in this enzymatic cocktail at 37°C without agitation for 10-13 minutes, with gentle pipetting every 5 minutes to facilitate tissue breakdown. The enzymatic reaction was stopped with ice-cold PBS, and cells were centrifuged at 300 RCF for 10 minutes and resuspended in ice-cold cell staining buffer (0.5% Bovine Serum Albumin, 2mM EDTA in PBS). Cells were stained with 1.25μL of anti-EpCAM-APC (Invitrogen 17-5791-82) antibody for 15 minutes at 4°C in the dark, followed by centrifugation and resuspension in FACS buffer (5% FBS, 5mM EDTA in PBS). To identify dead cells, DAPI was added to the cell suspension at a 1:1000 ratio. Cells were kept on ice and sorted using a Sony SH800S sorter, with gating strategies to exclude doublets and dead cells. DAPI negative, EpCAM positive cells were collected. RNA libraries were prepared according to 10X protocol.