SNAP25 protein, human

Nuclei were included for clustering analysis if they passed all of the following QC thresholds:
>30% cDNA longer than 400 base pairs
>500,000 reads aligned to exonic or intronic sequence
>40% of total reads aligned
>50% unique reads
TA nucleotide ratio > 0.7
After clustering (see below), clusters were identified as outliers if more than half of nuclei co-expressed markers of inhibitory (GAD1, GAD2) and excitatory (SLC17A7) neurons or were NeuN+ but did not express the pan-neuronal marker SNAP25. Median values of QC metrics listed above were calculated for each cluster and used to compute the median and inter-quartile range (IQR) of all cluster medians. Clusters were also identified as outliers if the cluster median QC metrics deviated by more than three times the IQRs from the median of all clusters. In total, 15,928 nuclei passed QC criteria and were split into three broad classes of cells (10,708 excitatory neurons, 4,297 inhibitory neurons, and 923 non-neuronal cells) based on NeuN staining and cell class marker gene expression
Clusters were identified as donor-specific if they included fewer nuclei sampled from donors than expected by chance. For each cluster, the expected proportion of nuclei from each donor was calculated based on the laminar composition of the cluster and laminar sampling of the donor. For example, if 30% of layer 3 nuclei were sampled from a donor, then a layer 3-enriched cluster should contain approximately 30% of nuclei from this donor. In contrast, if only layer 5 were sampled from a donor, then the expected sampling from this donor for a layer 1-enriched cluster was zero. If the difference between the observed and expected sampling was greater than 50% of the number of nuclei in the cluster, then the cluster was flagged as donor-specific and excluded. In total, 325 nuclei were assigned to donor-specific or outlier clusters that contained marginal quality nuclei and were excluded from further analysis. Three donor-specific clusters came from neurosurgical donors (n=95 nuclei) and were similar to other layer 5 types reported in our analysis, but had higher expression of activity-dependent genes.
To confirm exclusion, clusters automatically flagged as outliers or donor-specific were manually inspected for expression of broad cell class marker genes, mitochondrial genes related to quality, and known activity-dependent genes.

The details of the assay were published previously^{35 (link)}. Briefly, soluble rat neuronal SNARE complex containing
Syntaxin-1A (1–265, S249C, K253C), full-length SNAP-25 (1–206), and
Synaptobrevin-2 (1–96), was labeled with Oregon Green (forming covalent linkages
with two cysteine residues close to the C-terminus of Syntaxin-1A, and four native
cysteine residues of SNAP-25). The disassembly reactions were carried out using
FlexStation II (Molecular Devices) in a 384-well plate with a reaction volume of 60
µL. Each condition (different αSNAP mutants) was divided into four
replicas, and the average was plotted. A final concentration of 400 nM Oregon
Green-labeled SNARE complex, 2 µM αSNAP, and 85 nM NSF was included in the
reaction buffered with 50 mM TrisCl, pH 8.0, 20 mM NaCl, 2 mM ATP, 2 mM MgCl₂,
and 0.5 mM TCEP. To measure the initial SNARE complex disassembly rate, the first 20 data
points after adding the NSF were used for linear regression analysis, except for
αSNAP mutants D217A/E249K/E252K/E253K/ΔC, and K122E/K163E, for which the
first 1000 data points were used because the slope was close to zero. Note that the
disassembly rate of wild type proteins (Fig. 5f) is
comparable to that of previous experiments^{35 (link),39 (link)}, illustrating that our
purification method of NSF produces fully active protein.