title: GSM6668352: 7-2hr-Eto_rep1_R1.fastq.gz; Mus musculus; OTHER
library_construction_protocol: To dissociate cultured mouse cortical neurons for END-seq, Papain (Worthington Biochemical Cat: LK003178) was dissolved in TrypLE Express Enzyme solution at 37 °C for 20 minutes prior to sample collection. Culture media was gently aspirated and the cells were gently washed once with PBS at 37 °C. 500uL of papain solution was added to each well of neurons in 6 well plates and incubated at 37 °C for 1 min. Papain solution was removed via pipette and 500uL trituration solution (culture medium supplemented with freshly dissolved DNase) was added. Cells were gently triturated 5-10 times with wide bore pipette tip, transferred to conical tubes, and pelleted. Cells were gently resuspended in ice cold PBS containing 0.1% BSA and 0.5 mM EDTA for counting. Cells were stored on ice while an aliquot was quickly counted via hemocytometer. 1.5 million neurons for each experimental condition were added to a new conical tube and pelleted. Cells were then embedded in agarose plugs using Bio-Rad CHEF Mammalian Genomic DNA Plug Kit (Bio-Rad #1703591) and processed for END-seq as previously described19,90. Experimental groups were processed back-to-back, collecting cells in one treatment group and embedding them in agarose plugs before collecting cells in the next treatment group, in order to reduce over-digestion of the samples and to minimize time between cell dissociation and embedding in agarose. We observed better signal-to-noise in cells treated with etoposide (50uM). Etoposide was added to the cells 6 hours prior to collection, and was included at 50uM in all solutions applied to the cells until embedding in agarose. END-seq