GSM5238882: L1849-In_vivo_HEK239-dCas9-no_sgRNA-rep1; Homo sapiens; OTHER
For in vivo CasKAS experiments, HEK293T cells were seeded at 400,000 cells/well into a 6-well plate the day before RNP transfection. Media was exchanged 2 hours before transfection. For each well, 6,250 ng of Cas9 (MCLAB CAS9-200) or dCas9 (MCLAB dCAS9B-200) and 1,200 ng sgRNA was complexed with CRISPRMAX (Thermo Fisher CMAX00008) reagent in Opti-MEM (Thermo Fisher 51985091) following manufacturer's protocol. After incubation at room temperature for 15 minutes, the RNP solution was directly added to each well and gently mixed. The cells were incubated with the RNP complex for 14 hours at 37C. To harvest and perform kethoxal labeling, media was removed and room temperature 1X PBS was used to wash the cells. Cells were then dissociated with trypsin, trypsin was quenched with media, cells were pelleted at room temperature, and then resuspended in 100 uL of media supplemented with 5 мM N3-kethoxal (final concentration). Cells were incubated for 10 minutes at 37C with shaking at 500 rpm in a Thermomixer. Cells were then pelleted by centrifuging at 500 g for 5 minutes at 4C. Genomic DNA was then extracted using the Monarch gDNA Purification Kit (NEB T3010S) following the standard protocol but with elution using 85 uL 25 mM K3BO3 at pH 7.0. The click reaction was carried out by combining 175 uL purified and sheared DNA, 5 uL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 20 uL 10x PBS in a final volume of 200 uL or 87.5 uL purified and sheared DNA, 2.5 uL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 10 uL 10x PBS in a final volume of 100 uL. The reaction was incubated at 37C for 90 minutes. DNA was purified using AMPure XP beads (50 uL for a 100 uL reaction or 100 uL for a 200 uL reaction), beads were washed on a magnetic stand twice with 80\% EtOH, and eluted in 130 uL 25mM K3BO3. Purified DNA was then sheared on a Covaris E220 instrument down to ~150-400 bp size. For streptavidin pulldown of biotin-labeled DNA, 10 uL of 10 mg/mL Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602) were separated on a magnetic stand, then washed with 300 uL of 1x TWB (Tween Washing Buffer; 5 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 1 M NaCl; 0.05\% Tween 20). The beads were resuspended in 300 uL of 2x Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA; 2 M NaCl), the sonicated DNA was added (diluted to a final volume of 300 uL if necessary), and the beads were incubated for >15 minutes at room temperature on a rotator. After separation on a magnetic stand, the beads were washed with 300 uL of 1x TWB, and heated at 55C in a Thermomixer with shaking for 2 minutes. After removal of the supernatant on a magnetic stand, the TWB wash and 55C incubation were repeated.  Final libraries were prepared on beads using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645) as follows. End repair was carried out by resuspending beads in 50 uL 1x EB buffer, and adding 3 uL NEB Ultra End Repair Enzyme and 7 uL NEB Ultra End Repair Enzyme, followed by incubation at 20C for 30 minutes (in a Thermomixer, with shaking at 1,000 rpm) and then at 65C for 30 minutes. Adapters were ligated to DNA fragments by adding 30 uL Blunt Ligation mix, 1 uL Ligation Enhancer and 2.5 uL NEB Adapter, incubating at 20C for 20 minutes, adding 3 uL USER enzyme, and incubating at 37C for 15 minutes (in a Thermomixer, with shaking at 1,000 rpm) Beads were then separated on a magnetic stand, and washed with 300 uL TWB for 2 minutes at 55C, 1000 rpm in a Thermomixer. After separation on a magnetic stand, beads were washed in 100 uL 0.1X TE buffer, then resuspended in 15 uL 0.1X TE buffer, and heated at 98C for 10 minutes. For PCR, 5 uL of each of the i5 and i7 NEB Next sequencing adapters were added together with 25 uL 2X NEB Ultra PCR Mater Mix. PCR was carried out with a 98C incubation for 30 seconds and 12 cycles of 98C for 10 seconds, 65C for 30 seconds, and 72C for 1 minute, followed by incubation at 72C for 5 minutes. Beads were separated on a magnetic stand, and the supernatant was cleaned up using 1.8X AMPure XP beads. Libraries were sequenced in a paired-end format on a Illumina NextSeq instrument using NextSeq 500/550 high output kits (2x36 cycles).