GSM8522374: Cortex, s69; Homo sapiens; RNA-Seq
Nuclei extraction from snap-frozen human brains was performed on wet ice. The frozen, dissected tissue was lysed using a Dounce homogenizer (10 strokes with the loose pestle followed by 20 strokes with the tight pestle) in 600 μl of freshly made cold lysis buffer (low-sucrose buffer, Triton X-100 20%, RNase inhibitor 40 U μl−1). The homogenate was filtered through a 40-μm cell strainer for nuclei extraction with 4 ml of low-sucrose buffer (0.64 M sucrose, HEPES pH8, CaCl2, MgAc, EDTA, DTT and nuclease-free water). Sonication of the homogenate was performed for 10 s. To remove cellular and myelin debris, a sucrose gradient (12 ml of high-sucrose buffer (2 M sucrose, HEPES pH 8, MgAc, DTT, nuclease-free water) underneath the low-sucrose buffer) was centrifuged at 3200g for 30 min, and the supernatant was discarded. Nuclei were resuspended in 1 ml resuspension solution (PBS, 5% BSA and RNase inhibitor 40 U μl−1) and centrifuged for 12 min at 3200g. Nuclei were again resuspended in 50 μl cold resuspension solution. Nuclei were counted using trypan blue and a haemocytometer under bright field. 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