GSE136802
PESCA: A scalable platform for the development of cell-type-specific viral drivers
Highly paralleled functional evaluation of enhancer activity in single cells generates new cell-type-specific tools with broad medical and scientific applications. Overall design: For INTACT we crossed Sst-IRES-Cre (The Jackson Laboratory Stock # 013044), Vip-IRES-Cre (The Jackson Laboratory Stock # 010908) and Pv-Cre (The Jackson Laboratory Stock # 017320) with SUN1-2xsfGFP-6xMYC (The Jackson Laboratory Stock # 021039) and used adult (6-12 wk old) male and female F1 progeny. Nuclear purifications were performed from whole cortex of adult mice as previously described using anti-GFP antibodies (Fisher G10362). Isolated nuclei were gently resuspended in cold L1 buffer (50 mM Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.25% Triton X-100, 0.5% NP40, 10% Glycerol, protease inhibitors), and pelleted at 800g for 5 minutes at 4°C. DNA libraries were prepared from the nuclei using the Nextera DNA Library Prep Kit (Illumina) according to manufacturer's protocols.  The final libraries were purified using the Qiagen minelute kit and sequenced on a Nextseq 500 benchtop DNA sequencer (Illumina). 2 replicates were generated and sequenced for each mouse strain.     For PESCA screening we used adult (6-10 wk) C57BL/6J (The Jackson Laboratory, Stock # 000664) mice. Animals were anesthetized with isofluorane (1–3% in air) and placed on a stereotactic instrument (Kopf) with a 37°C heated pad. The PESCA library (AAV9, 1.9 x 1013 genome copies/mL) was stereotactically injected in V1 (800 nL per site at 25 nL/min) using a sharp glass pipette (25-45 µm diameter) that was left in place for 5 min prior to and 10 min following injection to minimize backflow. Two injections were performed per animal at coordinates 3.0 and 3.7 mm posterior, 2.5 mm lateral relative to bregma, and 0.6 mm ventral relative to the brain surface.  Individual rAAV-GRE constructs were stereotactically injected at a titer of 1 x 1011 genome copies/mL. (250 nL per site at 25 nL/min).  All injections were performed at two depths (0.4 and 0.7 mm ventral relative to the brain surface) to achieve broader infection across cortical layers. The injection coordinates relative to bregma were 3.0 or 3.7 mm posterior, 2.5 or -2.5 mm lateral. Single-nuclei suspensions were generated as described previously(1)  , with minor modifications. V1 was dissected and placed into a Dounce with homogenization buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, protease inhibitors). The sample was homogenized using a tight pestle with 10 stokes. IGEPAL solution (5%, Sigma) was added to a final concentration of 0.32%, and 5 additional strokes were performed. The homogenate was filtered through a 40-µm filter, and OptiPrep (Sigma) added to a final concentration of 25% iodixanol.  The sample was layered onto an iodixanol gradient and centrifuged at 10,000g for 18 minutes as previously described. Nuclei were collected between the 30% and 40% iodixanol layers and diluted to 80,000-100,000 nuclei/mL for encapsulation. All buffers contained 0.15% RNasin Plus RNase Inhibitor (Promega) and 0.04% BSA. Single nuclei were captured and barcoded whole-transcriptome libraries prepared using the inDrops platform as previously described(5, 6), collecting five libraries of approximately 3,000 nuclei from each animal. For enrichment of virally-derived transcripts, a fraction (3 µL) of the amplified RNA intermediate was reverse transcribed with random hexamers without prior fragmentation. PCR was next used to amplify virally derived transcripts. The forward primer was designed to introduce the R1 sequence and anneal to a sequence uniquely present 5' of the viral-barcode sequence present in the viral transcripts (5'- GCATCGATACCGAGCGC). The reverse primer was designed to anneal to a sequence present 5' of the cell-barcode (5'- GGGTGTCGGGTGCAG). The result of the PCR is preferential amplification of the viral-derived transcripts, while simultaneously retaining the cell-barcode sequence necessary to assign each transcript to a particular cell/nucleus. Following PCR amplification (18 cycles, Hot Start High-Fidelity Q5 polymerase) all the libraries were indexed, pooled, and sequenced on a Nextseq 500 benchtop DNA sequencer (Illumina).