GSM5643793: MEF; Mus musculus; RNA-Seq
Cultured cells were trypsinized, resuspended with PBS in single cell level, and used for 10X GEM generation. Single cell 3' GEM kit v3 (10xGenomics, Pleasanton, CA) were used for 10x library construction following the manufacturer's protocol. Briefly, cells were re-suspended in PBS as a concentration of 1000 cells/µl. Resuspended cells were loaded in chromium chip B as a recovery yield of ~10,000 with gel beads and oil. Generated GEM was used for cDNA amplification by PCR. Amplified cDNA then was fragmented, end repaired, and A-tailed. Adaptors was ligated, followed by index PCR and double sized selection. As a final step, the library was diluted in 10nM and used for sequencing. The sequencing was performed using Novaseq 6000 (Illumina, Inc, California, USA) with full SP flowcell. About 560 million reads were obtained for around 10,000 cells (over 40,000 reads per cells). 10x scRNA-Seq