title: GSM9024428: tdTomato,rep1; Mus musculus; RNA-Seq
library_name: GSM9024428
library_construction_protocol: Nuclei were isolated from frozen VC tissue. Tissue was manually homogenized using a two-step Dounce homogenizer (A and B) in NIMT buffer, containing (in mM: 250 sucrose, 25 KCl, 5 MgCl2, 10 Tris-Cl pH 8, 1 DTT; 1:100 dilution of Triton X100, Protease Inhibitor Cocktail; and 1:1000 dilution of RNaseOUT Recombinant Ribonuclease Inhibitor; SUPERase• In RNase Inhibitor [Thermo #AM2694]) on ice. Homogenized samples were mixed with 50% iodixanol (OptiPrep Density Gradient Medium;) and loaded onto 25% iodixanol cushion, and centrifuged at 10,000 g for 20 min at 4°C in a swinging bucket rotor. Pellets resuspended in ice-cold DPBS (HyClone) with 1:1000 dilution of RNaseOUT Recombinant Ribonuclease Inhibitor; SUPERase• In RNase Inhibitor. Nuclei were then incubated for 7 min on ice with Hoechst 33342 solution (20 mM)  (final concentration 0.5 µM), followed by centrifugation at 1000 g for 10 min at 4°C to pellet nuclei. Pellets were resuspended in blocking buffer containing DPBS with RNAse inhibitors, and 1:10 dilution of pure BSA, and blocked for 30 min on ice. Nuclei were purified using FACS Library was performed accoding to the manufacturer's instructions (10x genomics- single cell 3' v3.1 HT). Briefly, 50k sorted nuclei were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the GEMs(gel bead-in-emulsion). The RNA from each bead was retrotranscripted to cDNA, which contains the Illumina R1 primer sequence, unique molecular identifier (UMI) and the 10x barcode. the pooled barcoded cDNAwas cleaned up, amplified, and the appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During library construction the Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.