Nature research paper: A NPAS4–NuA4 complex couples synaptic activity to DNA repair
Nuclear isolation, fixation and adapter ligation
sBLISS-seq was carried out on cultured neuronal nuclei or on nuclei isolated from hippocampal tissue according to a previously described protocol. Replicates consisted of individual mice for the wild-type time course. For the Cre and ΔCre datasets, a replicate consisted of one infected hemisphere from one mouse. Samples were paired such that 2 h KA_CRE1 and 2 h KA_ΔCre1 came from the same mouse.
To process hippocampal tissue for sBLISS-seq, hippocampi were placed in 1 ml of buffer HB and dounced 5× with a loose pestle and 10× with a tight pestle. IGEPAL CA-630 was added before douncing with a tight pestle 5–8 more times and filtering through a 40-µm strainer. To preserve DNA breaks for the remainder of the protocol, nuclei were fixed with 2% PFA followed by quenching with 125 mM glycine. Fixed nuclei were gently pelleted by centrifuging at 500.
Note that the wild-type time course samples were performed in two batches with stimulation time points split across these two batches. We did observe a batch effect from processing the samples, which was computationally removed . The batch for the associated samples is listed in Supplementary Table. Note also that the wild-type time course samples included a 2% spike-in of human HEK293T cells expressing a guide-RNA-induced cut site.
To isolate nuclei from cultured neurons plated in 6-well dishes for Cas9 control experiments, neurons were washed with ice-cold PBS to remove debris and 1 ml of HB, and 32 µl of 5% IGEPAL CA-630 was added to each well. Cells were incubated for 10 min in HB with gentle rotation at 4 °C before removal by gentle scraping and transfer to Eppendorf tubes. Nuclei from cultured neurons were fixed with 2% PFA followed by quenching with 125 mM glycine and gently pelleted by centrifuging at 500.
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