GSM8453300: Alloxan (negative control, T=5), scRNAseq; Mus musculus; RNA-Seq
Pancreatic slices are washed 3x with dPBS transferred into 15ml of prewarmed (37C) Dissociation solution- Triple-E. 10 minutes incubation at 37C follows. Brief vortex 5seconds bursts repeated 10x and additional 2min. incubation. The vortex step is repeated one more time.  Dissociated slices are immediately washed in 20ml of wash medium and spun down at 1500 RPM. Washing is repeated and cells are put through 40-micron strainer. The single cells are now washed one time with dPBS at 4 C resuspended in 1ml dPBS and 10 µl of single cell suspension is taken to control  number and viability with Trypan blue. After additional spin, the pellet is resuspended in 200 µl of ice cold dPBS. 800 µl of ice cold methanol is added dropwise with constant stirring to prevent clumping during fixation. Fixed cells are kept in-20C for 24 hrs and later in -80C.  Single cell suspension is taken to control  number and viability with Trypan blue .After additional spin, the pellet is resuspended in 200 µl of ice cold dPBS. 800 µl of ice cold methanol is added dropwise with constant stirring to prevent clumping during fixation. Fixed cells are kept in-20C for 24 hrs and later in -80C. Slices are washed 3x with dPBS transferred into 15ml of prewarmed (37C) Dissociation solution- Triple-E. 10 minutes incubation at 37C follows. Brief vortex 5 seconds bursts repeated 10x and additional 2min. incubation. The vortex step is repeated one more time.  Dissociated slices are immediately washed in 20ml of wash medium and spun down at 1500 RPM. Washing is repeated and cells are put through 40-micron strainer. The single cells are now washed one time with dPBS at 4 C resuspended in 1ml dPBS and 10 µl of single cell suspension is taken to control  number and viability with Trypan blue. After additional spin, the pellet is resuspended in 200 µl of ice cold dPBS. 800 µl of ice cold methanol is added dropwise with constant stirring to prevent clumping during fixation. Fixed cells are kept in-200C for 24 hrs and later in -80 C. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.