GSM7885459: WT P1 Maxillary Molar; Mus musculus; RNA-Seq
E15.5 embryos and P0/P1 mice pups (n=6) were used for single-cell dissociation. Mice embryos/pups were dissected out and transferred into ice-cold phosphate-buffered saline (PBS) in a 10 cm petri dish. Tooth organs were carefully dissected (maxillary and mandibular molars) of each respective stage and were collected in a 1.5 ml tube with 1X PBS with 0.04% bovine serum albumin (BSA) on ice. Samples were collected from mice embryos/pups in each respective age group. Harvested tooth organs were cut into small pieces in ice-cold 1X PBS with 0.04% BSA on ice. For isolating the single cells, Tissue pellets were then incubated in 1 ml of TrypLE express enzyme (Thermo Fisher Scientific, Waltham, MA) for 30 minutes at 37℃. Cells were collected by passing the suspension through a cell strainer, followed by centrifugation. Libraries from isolated cells were prepared using the Chromium Next Single Cell 3' Reagent Kit v3.1 (Dual Index -10x Genomics – protocol CG000315 Rev C), aiming for a total of 8,000 cells per sample. In brief, cell suspensions were loaded onto the Chromium Controller microfluidic device to be partitioned into bar code-containing Gel Bead-In-Emulsions (GEMs). Samples were incubated, and poly-adenylated mRNAs were reverse-transcribed into cDNAs containing a unique molecular identifier (UMI) and a cell barcode unique to each GEM. The GEMs were broken using a recovery agent to produce a bulk pool of barcoded molecules. Additional amplification of the cDNA was performed before proceeding to fragmentation, end repair, A-tailing, and ligation. A sample dual index PCR was performed in order to add the P5, i5, i7, and P7 sequences to the final scRNA-seq library. Finally, samples were pooled, and the library was sequenced using the Illumina NovaSeq 6000 with the SP reagent Kit v.1.5, resulting in 28x90 bp paired-end reads.