GSM5115980: TBI and Control; Mus musculus; RNA-Seq
Frozen brain tissue was thawed in 500 uL Buffer HB (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH pH 7.8, 0.15mM spermine tetrahydrochloride, 0.5mM spermidine trihydrochloride, 1mM DTT). The tissue was transferred to a 2 mL dounce. 500ul 5% IGEPAL CA-630 (Sigma) and 1 mL HB were added to the tissue and the tissue was homogenized with a tight pestle 10-15 times. The sample was transferred to a 15 mL tube and total solution brought to 9 mL with HB. In a Corex tube (Fisher Scientific), 1 mL 30% iodixanol layered on top of 1 mL 40% iodixanol. The 9 mL sample was layered on top of the iodixanol cushion. The sample was spun at 10,000 xg for 18 minutes. 1 mL of sample at the 30-40% iodixanol interface was collected. After counting nuclear with a hemocytometer, the sample was diluted to 100,000 nuclear/mL with 30% iodixanol (with RNAsin) and subjected to single nuclear droplet encapsulation with inDrops. Individual nuclei were captured and barcoded using the inDrops platform as previously described.1 Briefly, single-cell suspensions were fed into a microfluidic device that packaged the cells with barcoded hydrogel microspheres and reverse transcriptase/lysis reagents. After cell encapsulation, primers were photo-released by UV exposure. Two libraries of approximately 3000 cells each were collected for each sample. Indexed libraries were pooled and sequenced on a Nextseq 500 (Illumina).