Instrument: NextSeq 500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: In vitro CRISPR: Cells were detached and resuspended in basic culture media (media excluding growth factors) with propidium iodide (1:1,000; BD Biosciences) and strained (70 mm filters, BD Biosciences). RFP+ cells were FACS isolated at 4°C (reanalysis showed >99% purity) and pelleted at 1,500 rpm for 5 min, snap frozen on dry ice and stored at -80°C until RNA/DNA were isolated. For all in vivo CRISPR experiments animals were sacrificed after two months and analyzed either by IHC or nuclei isolation (see details below) followed by DNA- or RNA-seq. Emx animals (RNAseq): Animals were sacrificed at 3 months of age for either immunohistochemistry IHC or RNA sequencing. Emx animals (single cell): The nuclei isolation was performed according to Sodersten et al. (2018). The tissue was thawed and dissociated in ice-cold lysis buffer ((0.32 M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM DTT) using a 1 ml tissue douncer (Wheaton). The homogenate was carefully layered on top of a sucrose cushion (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl, pH 8.0, and 1 1 mM DTT) before centrifugation at 30,000 × g for 2 hours and 15 min. Pelleted nuclei were softened for 10 minutes in 100 ml of nuclear storage buffer (15% sucrose, 10 mM Tris-_HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) before resuspended in 300 ml of dilution buffer (10 mM Tris-_HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) and run through a cell strainer (70 mm). Cells were run through the FACS (FACS Aria, BD Biosciences) at 4°C with low flowrate using a 100 mm nozzle (reanalysis showed >99% purity). RNAseq: Total RNA was isolated from frozen cell/nuclei pellets and brain tissue using the RNeasy Mini Kit (Qiagen) and used for RNA-seq and qPCR (tissue pieces were run in the tissue lyser for 2 min, 30 Hz, prior to RNA isolation). Single cell: 8,500 nuclei per sample were sorted via FACS and loaded onto 10X Genomics Single Cell 3' Chip along with the reverse transcription mastermix following the manufacturer's protocol for the Chromium Single Cell 3′ Library (10X Genomics, PN-120233) to generate single-cell gel beads in emulsion. cDNA amplification was done as per the guidelines from 10x Genomics and sequencing libraries were generated with unique sample indices (SI) for each sample. RNAseq libraries were generated using Illumina TruSeq Stranded mRNA library prep kit (poly-A selection) and sequenced on a NextSeq500 (PE 2 × 150bp). Cut and run (IgG and H3K9me3) library preparation was performed using the Hyper prep kit (KAPA biosystems) and sequenced on NextSeq500 2 x 75 bp. For the single cell sequencing, libraries for samples were multiplexed and sequenced on a Novaseq using a 150-cycle kit.