single-cell whole genome sequencing through PTA amplification for donor 6052; library 2
Single neuronal nuclei, prepared as described above, were whole-genome amplified by primary template-directed Amplification (PTA) using the ResolveDNA Whole Genome Amplification Kit (BioSkryb Genomics). First, nuclei were sorted into cold 96-well plates pre-loaded with 3µl cold Cell Buffer (BioSkryb) one-per-well. Nuclei were lysed per kit protocol by addition of 3 µl MS mix followed by a brief spin-down, then 1 minute of agitation at room temperature at 1,400 RPM on a plate mixer, then 10 minutes on ice. Next. 3ul SN1 buffer was added to each well and the plate was again spun down and agitated at 1,400 RPM for 1 minute. Next, 3µl SDX buffer was added, and the plate was again spun and agitated at 1,400 rpm for 1 minute. Then, the plate was incubated at room temperature for 10 minutes. Next reaction mix + enzyme were added to each well, for a total reaction volume of 20 µl/well. PTA was carried out for 10 hours at 30 °C, followed by enzyme inactivation at 65 °C for 3 min. Amplified DNA was cleaned up using an in-house carboxyl magnetic bead cleanup solution (0.024M PEG-8000, 1M NaCl, 1mM EDTA, 10mM Tris-HCl pH 8, 0.055% Tween 20, and 1.5ml Cytiva Sera-Mag SpeedBeads™ Carboxyl Magnetic Beads, hydrophobic per 50 ml). DNA yield was determined using the QuantiFluor dsDNA System (Promega). Samples were subjected to quality control by multiplex PCR for four genomic loci on different chromosomes as previously described. Amplified genomes showing positive amplification for all four multiplex PCR loci were prepared for Illumina sequencing. Libraries were prepared following a modified KAPA HyperPlus Library Preparation protocol described in the ResolveDNA EA Whole Genome Amplification protocol. In brief, the fragmentation step was skipped and end repair + A-tailing was performed for 500 ng amplified DNA input. Adapter ligation was then performed using the SeqCap Adapter Kit (Roche, 07141548001). Ligated DNA was cleaned up using in-house beads and amplified through an on-bead PCR amplification step. Amplified libraries were selected for a size of 300-600 bp using double-size selection. Libraries were subjected to in-house quality control using a 5300 Fragment Analyzer Bioanalyzer for DNA fragment size distribution (Agilent Technologies). Successfully prepped samples were sent to Genuity Sciences (Dublin, Ireland) for DNA sequencing, who further tested for quality using TapeStation (Agilent Technologies) prior to processing. Single-cell PTA-amplified genome libraries were sequenced on the Illumina NovaSeq 6000 platform (150 bp × 2) at minimum 20× coverage (Table S12). scWGS of some neurons was performed at Harvard for previous publications (Table S12).