GSM8508907: CT_scRNA; Mus musculus; RNA-Seq
An appropriate amount of human cardiac septal tissue was taken and placed into the serum-free medium with sterile forceps, and the tissue was minced with sterile scissors and filtered through a 40 μm filter to obtain the cell suspension. Subsequently, an appropriate amount of type II collagenase was added, and digestion was carried out in a 37 ℃ water bath for 20 min to terminate the enzyme digestion, and the residual collagenase was washed away by centrifugation-discarding the supernatant and resuspending with culture medium. 300g-500g for 5 min-8 min to remove the cellular debris. Finally, erythrocyte lysis was performed using erythrocyte lysis solution. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, CM cells were resuspended in the master mix and loaded together with partioning oil and gel beads into the chip to generate the gel bead-n-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single was retrotranscripted cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UM) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropriated sized fragments were selected with SPRIseclect reagent for subsequent library construction. During the library construction Illumina R2 primer R2 sequence, paried-end constructs with P5 and P7 sequences and a sample index were added.