D1000 screentape assay

For each PCR, 3 µl of DNA solution was electrophoresed in a 2% agarose gel to check amplification efficiency. SSU rDNA and mini-COI libraries were prepared separately. Next, the amplicons were pooled and purified using a 2% E-Gel SizeSelect II Agarose Gel System (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s protocol. The DNA concentration and fragment length distribution of the libraries were determined using a High-Sensitivity D1000 Screen Tape assay on a 2200 Tape Station system (Agilent Technologies, Inc., Santa Clara, CA, USA). Clonal template amplifications were performed using the Ion Torrent One Touch System II and Ion Torrent OT2 Kit (Life Technologies, Thermo Fisher Scientific) according to the manufacturer’s protocol. For emulsion PCR, SSU rDNA and mini-COI libraries were pooled in a 10:1 ratio. Sequencing was performed using the Ion 540 Kit-OT2 and Ion S5 systems on Ion 540 chips (Life Technologies, Thermo Fisher Scientific), according to the manufacturer’s instructions. Sequencing was designed to yield approximately 10,000 and 1000 reads per SSU and COI amplicons, respectively.

GFP+ cells from ~500 tails were collected for single cell analysis using the above methods but scaled for 500 tails. After sorting, cells were immediately washed with 1xPBS and diluted to a concentration of 1000 cells/µl based on the 10x Genomics guidelines. We aimed for capture of 10,000 cells using the 10x Genomics Platform. Single-cell mRNA libraries were prepared using the single-cell 3’ solution V2 kit (10x Genomics). Quality control and quantification assays were performed using a Qubit fluorometer (Thermo Fisher) and a D1000 Screentape Assay (Agilent). Libraries were sequences on an Illumina NextSeq 500 using 75-cycle, high output kits (reads 1: 26 cycles, i7 Index: eight cycles, read 2: 57 cycles). Each sample was sequenced to average read depth of 16 million total reads. This resulted in an average read depth of ~15,000 reads/cell after read depth normalization.

The 18S rDNA and mini-COI libraries were prepared separately. For each PCR reaction, 3 µl was electrophoresed on a 2% agarose gel to check amplification efficiency. Then, the amplicons were pooled and purified using 2% E-Gel SizeSelect II Agarose Gels system (Invitrogen, USA), according to the manufacturer’s protocol. DNA concentration and fragment length distribution of the libraries were established using High Sensitivity D1000 Screen Tape assay on 2200 Tape Station system (Agilent Technologies, USA). For the emulsion PCR, the 18S rDNA and mini-COI libraries were pooled in the 10:1 ratio. Emulsion PCR and sequencing were carried out using the Ion Torrent One Touch System II, the Ion 520 and Ion 530 Kit-OT2, Ion 530 chip and the S5 system (Life Technologies) according to the manufacturer's instructions.

ATAC-seq sample preparation was performed for eight biological replicates/conditions according to detailed protocols obtained from Hongjun Song’s lab at the University of Pennsylvania (Buenrostro et al., 2015 (link); Su et al., 2017 (link)). In brief, one hippocampus was homogenized with a Dounce tissue grinder (Wheaton 357544) in 2 mL HB buffer (1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, protease inhibitor (Sigma-Aldrich 04693159001), 0.3% IGEPAL-630, 0.25 M sucrose, 25 mM MgCl₂, 20 mM Tricine-KOH). The homogenate was filtered through a 40-μm strainer and centrifuged over one volume of cushion buffer (0.5 mM MgCl₂, 0.5 mM DTT, protease inhibitor, 0.88 M sucrose) at 2,800g for 10 min in a swinging bucket centrifuge at 4°C. The nuclei pellet was resuspended in 20 μL PBS. Then, 1 μL of the nuclei sample was stained with Hoechst 33342 to calculate nuclei concentration, and 50,000 nuclei per sample were used for downstream library preparation. Libraries were prepared using the Nextera DNA library prep kit (Illumina FC-121-1030), and quality was analyzed using the D1000 ScreenTape Assay (Agilent 5067-5582). Paired-end 50 bp sequencing to a depth of ~40 million reads per sample was performed using the Illumina HiSeq 2500 system at the UCLA Broad Stem Cell Research Center Sequencing Core.