GSM6697776: E12.5DLX2; Mus musculus; RNA-Seq
Fresh maxillary prominence tissues were conserved in the GEXSCOPE® Tissue Preservation Solution (Singleron) until library preparation. scRNA-seq libraries were constructed using a microwell-based system (Zhang K, et al., Nucleic Acids Research, 2018). Briefly, the specimens were washed 3 times with Hanks Balanced Salt Solution (HBSS, Gibco, Cat. No.14025-076), shredded into 1–2 mm pieces, and then digested with 2ml GEXSCOPE® Tissue Dissociation Solution (Singleron) at 37℃ for 15min in a 15ml centrifuge tube (Falcon, Cat. No.352095) with sustained agitation. After digestion, cells were filtered through 40-micron sterile strainers (Falcon, Cat. No.352340) and centrifuged (Eppendorf, 5810R) at 1,000rpm for 5 minutes. The supernatant was removed, and the pellets were resuspended in 1ml PBS (Hyclone, Cat. No.SA30256.01). To remove the red blood cells, 2 mL RBC lysis buﬀer (Roche, Cat. No. 11 814 389 001) was added to the cell suspension according to the manufacturer's protocol. Cells were collected by centrifugation at 500g for 5 min and resuspend in PBS (Hyclone, Cat. No.SA30256.01). An aliquot from the cell mixture was stained with trypan blue (Bio-RAD, Cat. No.#1450013) and counted on an ECLIPSE Ts2 cellcounter (Nikon). The concentration of the single-cell suspension was adjusted to 1×105 cells/mL using PBS. Single-cell suspensions were then loaded onto microfluidic devices and scRNA-seq libraries were constructed according to Singleron GEXSCOPETM protocol by GEXSCOPETM Single-Cell RNA Library Kit (Singleron Biotechnologies). Individual libraries were diluted to 4nM and pooled for sequencing. Pools were sequenced on Illumina HiSeq X with 150 bp paired end reads.