title: GSM2943544: Macx; Mus musculus; RNA-Seq
library_construction_protocol: Protocol was adapted from (Halder et al., 2015). Mice were euthanized by CO2 inhalation, the lumbar spinal cord was then rapidly dissected, and the dorsal root ganglia removed. Cords were dounced in 500ul sucrose buffer (0.32M sucrose, 10mM HEPES pH 8.0, 5mM CaCl2, 3mM Mg-Acetate, 0.1mM EDTA, 1mM DTT) with 0.1% Triton X-100 using five strokes with the “A” pestle (Kontes Dounce Tissue Grinder), followed by five strokes with the “B” pestle. The lysate was then diluted with 3 mL sucrose buffer and was centrifuged at 3,200xg for 10 minutes. The supernatant was removed and 3 mL of sucrose buffer was added to the pellet and incubated for 1-2 minutes and the loosened pellet was then transferred to an Oak Ridge centrifuge tube. The pellet was then homogenized using an Ultra-Turrax on setting “1” for one minute. 12mL of density buffer (1M sucrose, 10mM HEPES pH 8.0, 3mM Mg-Acetate, 1mM DTT) was then added carefully below the nuclei layer and the tube was centrifuged at 3,200xg for 20 minutes. The supernatant was then rapidly poured off and the nuclei on the walls of the tube were collected with 1mL of PBS with 0.01% BSA and spun at 3,200xg for 10 minutes. Nuclei were then resuspended in PBS with 0.01% BSA. Drop-Seq was performed as previously described (Macosko et al., 2015) with the following modifications. Nuclei in 1mL of PBS with 0.01% BSA were counted with a hemocytometer and adjusted to a final concentration of 225 nuclei/ul. Beads were prepared at a concentration of 250 beads/ul in a lysis buffer that contained 0.7% sarkosyl. The following flow rates were used: beads (35ul/min), nuclei (35ul/min), oil (200ul/min). Ten PCR reactions were performed for each sample, and these were pooled, purified with AMPure beads (0.6X), quantified with Qubit 3.0, and analyzed on a High Sensitivity DNA Bioanalyzer chip. Multiple tagmentation and library preparation reactions were performed for each sample, each with 600pg input. Parallel reactions were then pooled, double purified with AMPure beads (0.8X), quantified with Qubit 3.0, and analyzed on a High Sensitivity DNA Bioanalyzer chip. Libraries were sequenced at a minimum depth of 20,000 reads per nucleus using HiSeq 2500 Rapid Mode with the following parameters: Read 1 25bp, index, custom primer; Read 2 50bp, Illumina primer. Each sample was produced from the lumbar cords of a pair of ICR mice, ages 8-11 weeks old. There were six independent samples for baseline, prepared on four different days (three male and three female), five independent samples for formalin treatment, prepared on three different days (three male and two female), and five independent samples for rotarod behavior, prepared on two different days (three male and two female). For all formalin samples, a second round of sequencing was performed, producing ten sequencing reactions for the five biological replicates.
attributes.tag: GEO Accession
attributes.value: GSM2943544