5 million murine Neuro2a cells were transfected with 10QATXN7 or 92Q-ATXN7 and co-transfected with a plasmid expressing sgRNA to a designated target site on Chromosome 14 (site 1: GGTAGACATCAGCGCTGTTG, site 2: CTCGCAGCCCTCCACCGTAA), along with CRISPR-Cas9 and a constitutive GFP reporter.  For the quality control experiment, only the CRISPR-Cas9 plasmid co-expressing site 2 targeted sgRNA or non-targeted sgRNA were used.  48 hrs after transfection, cells were trypsinized for 5 min, rinsed in PBS, and three replicates were pooled. 1 million GFP positive cells per pooled condition were sorted by flow cytometry.  Cells were then resuspended in 200 μl PBS and 20 μl NEB Proteinase K (800U/mL) and incubated for 30 min at 56°C.  Proteinase K was inactivated by heating at 95°C for 5 min and then 5 µl RNaseA (8mg/ml) was added and incubated for 30 min at 37°C.  Treated lysate was purified using the Qiagen Blood and Tissue DNA Kit using the manufacturer's protocol, with the final elution performed twice with 100 µl AE buffer. A Diagenode Bioruptor Pico was used to shear DNA to 750 bp fragments.  Each sample was then bait-amplified using using Phusion DNA polymerase (ThermoFisher #F530L) in standard reactions containing biotinylated bait primers (site 1: /5Biosg/ACTAAAAAGAGCGCCCCTCTTTC, site 2: /5Biosg/GGTTTCCCCTCCGCTCTTTA). The Invitrogen PureLink PCR Cleanup Kit was used to remove excess RNA and biotinylated primer.  50 µl 5M NaCl and 2.5 µl 0.5M EDTA pH 8 were added to each sample.  Biotinylated bait DNA was then purified using magnetic C1 Streptavidin Dynabeads (ThermoFisher #11205D).  Bead-DNA complexes were washed and resuspended in water and then the following was added for an on-bead ligation reaction: 10 µl 10X T4 ligase buffer, 5 µl 20mM hexamine cobalt III chloride (Sigma #481521), 5 µl bridge adapters as per reference, 5 µl3U/µl T4 DNA ligase (NEB #M0202S), and 30 µl 50% PEG8000 (Promega #V3011) in water.  A thermal cycler was used for the ligation reaction using settings of 1 hr at 25°C, 1 hr at 22°C, a manual pipette resuspension step, 1 hr at 22°C, and 16 hrs at 16°C.  Ligation product was then washed by first adding 100 µl 2X B&W buffer per sample, captured on magnets, and then washing with 200 µl 1X B&W buffer twice followed by a 200 µl water wash.  LpnPI digestion was performed on indicated samples using beads resuspended in 23.5 µl water, 3 µl 10X NEB Cutsmart buffer, 0.5 µl of 100 µM 5-Cytosine methylated primer (site 1: TTTGGGAAGAGC/iMe-dC/TGAGCATTCTGAG, site 2: TCAACCCCGTCCC/iMedC/AGTGGAAAACACCC), 0.5µl LpnPI enzyme (NEB #R0663S), 1 µl 30X enzyme activator, at  running conditions of 37°C for 4 hrs, then 65°C heat inactivation for 20 min.  LpnPI digested  samples were washed with 600 µl B&W buffer 5x, then once with 1 mL water, and resuspended in 200 µl water. For Illumina adapter inclusion and barcoding, resuspended samples were split into 8 PCR reactions consisting of 10 µl 5X Phusion buffer, 4 µl 2.5mM dNTPs, 2 µl 10µM I5 nested primer, 2 µl 10µM I7 primer, 0.5 µl Phusion polymerase, 25 µl bead-DNA complex, and 6.5 µl water. Reaction was run using 95°C 30 sec, 18 cycles of 95°C 15 sec, 60°C 30 sec, 72°C 1 min, then 72°C 6min, and ending with 4°C until removed from thermal cycler.  PCR products were pooled and   removed of beads by extraction of supernatant on magnet followed by centrifugation at 15,000g for 5 min at RT.  Supernatant was transferred to a new tube and purified using the Invitrogen PureLink PCR Cleanup Kit with Buffer 2 conditions and eluted with 60 µl water.  Final Illumina binding adapters were added by 4 PCR reactions of P5-I5 and P7-I7 adapter primers, using the following conditions: 15 µl DNA products, 16.5 µl water, 10 µl 5X Phusion buffer, 4 µl 2.5mM dNTPs, 2 µl of each 10 µM P5-I5 and P7-I7 primer, 0.5 µl Phusion polymerase, with running conditions of 95°C 3min, 16 cycles of 95°C 15 sec, 62°C 30 sec, 72°C 1 min, then 72°C 6 min, and 4°C until removal from thermal cycler.  Final purification of library prior to NGS steps was performed by running on a 1X TBE gel containing GelRed stain (VWR #89139-134), excised in the 350-1,250bp range (directly above primer-dimer band), and extracted using a Qiagen gel purification kit.  Additional purification was performed from the gel purification by a second purification through the Invitrogen PureLink PCR Cleanup Kit with Buffer 2 conditions and eluted with 30 µl water.  Library concentration was estimated using a NanoDrop, and library pools were created using equimolar DNA per sample.  Finally, libraries were run on an Illumina MiSeq at 300 bp depth (QC reads, P5-I5-end only) or 250bp depth (experimental reads, P5-I5 end only).