GSM9312043: E13.5 G2G3W1 MNP; Mus musculus; RNA-Seq
E13.5 mandibles from control CD1 or Gli2f/f;Gli3f/f;Wnt1-Cre mouse embryos were dissected in ice-cold DEPC PBS and minced to a fine paste. 12.5 mg of tissue was placed in a sterile 1.5 mL tube containing 0.5 mL protease solution containing 125 U/mL DNase and 5 mg/mL Bacillus Licheniformis. The samples were incubated at 4C for a total of 10 minutes, with trituration using a wide boar pipette tip every minute after the first two. Protease was inactivated using ice-cold PBS containing 0.02% BSA and filtered using 30 mM filter. The cells were pelleted by centrifugation at 200g for 4 minutes and resuspended in 0.02% BSA in PBS. Cell number and viability were assessed using a hemocytometer and trypan blue staining. 9,600 cells were loaded onto a well on a 10X Chromium Single-Cell instrument (10X Genomics) to target sequencing of 6,000 cells. Barcoding, cDNA amplification, and library construction were performed using the Chromium Single-Cell 3' Library and Gel Bead Kit v3. Post cDNA amplification and cleanup was performed using SPRI select reagent (Beckman Coulter, Cat# B23318). Post cDNA amplification and post library construction quality control was performed using the Agilent Bioanalyzer High Sensitivity kit (Agilent 5067–4626). Libraries were sequenced using a NovaSeq 6000 and the S2 flow cell. Sequencing parameters used were: Read 1, 28 cycles; Index i7, eight cycles; Read 2, 91 cycles, producing about 300 million reads.