GSM7372104: Male Duodenum replicate 2, snRNAseq; Mus musculus; RNA-Seq
We used CitraPrep to isolate nucle from high degradative tissues. Frozen tissue was placed into a petri dish on ice and cut into 0.2cm cubed pieces or smaller using clean scissors and forceps in 1mL of CitraHB (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH pH 7.8, 10mM citric acid, 0.5U/uL RNasin, 0.5U/uL SUPERase RNase inhibitor, 1mM DTT, 0.15mM spermine tetrahydrochloride, 0.5mM spermidine trihydrochloride, and cOmplete, EDTA-free protease inhibitor). Chopped tissue (totaling less than 1.5cm cubed) was transferred to the glass dounce homogenizer on ice and 4mL of CitraHB was added. Tissue was incubated on ice for 7 minutes. Tissue was homogenized 20x with loose pestle before adding 320uL of NP-40 and homogenizing 40x with tight pestle. If tissue does not homogenize, an electric homogenizer with short bursts may be used. Nuclei were observed under the microscope for quality control before proceeding. Tissue lysate was filtered through a 40um strainer into a 50mL conical tube. Next, 5mL of 50% iodixanol solution (5 volumes OptiPrep and 1 volume of Diluent composed of 150mM KCl, 30mM MgCl2, 120mM Tricine-KOH pH 7.8 mixed, plus 10mM citric acid, 0.5U/uL RNasin, 0.5U/uL SUPERase RNase inhibitor, 1mM DTT, 0.15mM spermine, and 0.5mM spermidine) was added to the filtered tissue lysate to make a 25% iodixanol solution and vortexed to mix. The tissue lysate was slowly underlaid with 30% iodixanol solution (30% OptiPrep in CitraHB with 0.5U/uL RNasin, 0.5U/uL SUPERase RNase inhibitor, 1mM DTT, 0.15mM spermine, and 0.5mM spermidine), then slowly underlaid with 40% iodixanol solution (40% OptiPrep in CitraHB with 0.5U/uL RNasin, 0.5U/uL SUPERase RNase inhibitor, 1mM DTT, 0.15mM spermine, and 0.5mM spermidine). Without disturbing the layers, the 50mL tube was weighed and balanced before centrifugation at 10,000g for 18 minutes at 4oC with no brake. Nuclei were collected from the interface layer with a 1000mL pipette and placed into a new tube with 10mL nuclei wash buffer (1x PBS, 1% BSA, 0.5U/uL RNasin, and 0.5U/uL SUPERase RNase inhibitor) and vortexed before filtering through a 20um filter. Nuclei were pelleted by centrifugation at 500g for 5 minutes at 4oC. Supernatant was removed and tissue was washed a second time. The pellet was resuspended in 250uL of nuclei wash buffer and nuclei were counted using a hemocytometer. Library was performed according to the manufacter's instructions (single cell 3' GEM v3.1 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.