title: GSM5945062: Specimen_35; Homo sapiens; OTHER
library_construction_protocol: Tissue were minced with a razor blade and homogenized using a tissue grinder in a 1 ml of HB buffer (1 mM DTT), 0.15 mM spermine, 0.5 mM spermidine, EDTA-free protease inhibitor, 0.3% IGEPAL-630, 0.25 M sucrose, 25 mM MgCl2, 20 mM Tricine-KOH) for 5 to 10 strokes, then filtered through a 40 mm strainer, underlayer with a cushion buffer (0.5 mM MgCl2, 0.5 mM DTT, EDTA-free protease inhibitor, 0.88 M sucrose) and centrifuged at 2800g for 10 minutes in a swinging bucket centrifuge at 4 ºC. Nuclei were collected as pellets. Nuclei were then centrifuged for 3 mins at 500g at 4 ˚C. The pellet was resuspended in 1 ml of cold PBS-RI (1x PBS + 0.05U/μl RNase Inhibitor). The nuclei were passed through a 40 μm strainer. 3 ml of cold 1.33% formaldehyde solution was then added to 1 ml of cells. Nuclei were fixed for 10 mins before adding 160 μl of 5% Triton X-100. Nuclei were then permeabilized for 3 mins and centrifuged at 500g for 3 mins at 4 ˚C. Nuclei were resuspended in 500 l of PBS-RI before adding 500 μl of cold 100 mM Tris-HCl pH 8. Then, nuclei were spun down at 500g for 3 mins at 4 ˚C and resuspended in 300 μl of cold 0.5 X PBS-RI. Finally, nuclei were again passed through a 40 μm strainer and then counted on a hemocytometer, diluted to 1,000,000 cells/ml. mRNA from single nuclei were tagged 3 rounds with barcoded primers, with in-cell ligations using T4 DNA ligase. Plates were incubated for 30 mins at 37 ºC with gentle sharking (50 rpm) to allow hybridization and ligation to occur. The ligation products were purified with Dynabeads MyOne Streptavidin C1 beads. After washing beads once with 10 mM Tris and 0.1% Tween-20 solution and once with water, beads were resuspended into a solution containing 110 μl of 2X Kapa HiFi HotStart Master Mix, 8.8 μl of 10 μM stocks of primers BC_0062 and BC_0108, and 92.4 μl of water. PCR thermocycling was performed as follows: 95 °C for 3 mins, then five cycles at 98 °C for 20 seconds, 65 °C for 45 seconds, 72 °C for 3 mins. After these five cycles, Dynabeads beads were removed from PCR solution and EvaGreen dye was added at a 1X concentration. Samples were again placed in a qPCR machine with the following thermocycling conditions: 95 °C for 3 mins, cycling at 98 °C for 20 seconds, 65 °C for 20 seconds, and then 72 °C for 3 mins, followed by a single 5 mins at 72 °C after cycling. Once the qPCR signal began to plateau, reactions were removed. 