GSM7290910: kidney papilla, K2200020; Homo sapiens; RNA-Seq
7 sections (40um) of Human kidney sample were put into 1.5 ml tube on dry ice, then on wet ice, add 1 ml of ice cold NEB/PIC buffer with RNase inhibitor. After pipette sections up and down using a P1000 tip (cut the end off to increase pore size) to disperse and dissolve OCT, then using regular P1000 tip, pipette to further dissociate tissues into manageable sizes. Transfer solution to Dounce homogenizer, gently dounce tissue on ice: (with pestle A first, followed with pestle B). Transfer dounced solution to a 15 ml tube. Wash dounce with 1 ml of ice cold NEB/PIC buffer with RNase inhibitor, and then add this into the same tube. Incubate on ice for 10 min. Pass supernatant through 30 um CellTrics filter to a new 15 ml tube, bring up to 10 ml with PBSE/PIC with RNase inhibitor. Spin at 900xg for 10 min at 4 0C to pellet nuclei, then remove the supernatant, re-suspend the pellet in 20 ul of 1X Nuclei buffer. After count nuclei using hemocytometer, adjust the nuclei concentration to 3100-5000 nuclei/ul. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs 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. snRNA-seq