GSM8125082: Parental-nocrRNA; Homo sapiens; OTHER
Genomic DNA was harvested from 20 million cells using the Qiagen Genomic Tips Kit (cat #10243). We used a custom protocol adapted from the Nanopore Cas9 Sequencing Kit user's manual (SQK-CS9109, though this kit was not actually used) to enrich for genomic DNA surrounding crRNA target sites for Nanopore sequencing using Kit 14 chemistry. Except where specified below, reagents are from the Nanopore Ligation Sequencing Kit V14 (SQK-LSK114). To summarize in brief, synthetic spCas9 Alt-R crRNAs targeting ~15-20kb regions surrounding target sites of crCD55-4, crCD81-1, crKIT-2, crKIT-3, crB2M-1, and crB2M3 were designed according to instructions for the Nanopore Cas9 Sequencing Kit and ordered from IDT. At least two guides were used to target the (+) strand upstream of the Cas12a crRNA target sites, and at least two guides were used to target the (-) strand downstream of the target sites. Synthetic spCas9 guides were pooled and annealed with spCas9 tracrRNA (IDT Cat # 1072532) by heating to 95deg. C for 5min and cooling on the bench at room temperature, followed by assembly into ribonucleoprotein complexes by incubating the following mixture at room temperature for 30min: 10ul annealed crRNA:tracrRNA pool (100uM), 10ul 10x NEB rCutSmart Buffer (NEB B6004S), 79.2ul nuclease-free water, and 0.8ul HiFi Cas9 (62uM, S. pyogenes HiFi Cas9 nuclease V3 IDT Cat#1081060). 5ug of genomic DNA was dephosphorylated in a 30ul reaction volume using 15U of Quick calf intestinal phosphatase (NEB cat #M0525) and 3ul of 10x rCutsmart Buffer (NEB B6004S), incubated for 37deg. C for 10min, 80deg. C for 2min, and hold at 20deg. C. The entire volume of dephosphorylated genomic DNA is then mixed with 10ul of Cas9 ribonucleoprotein, 1ul 10mM dATP, and 1ul Taq polymerase for a total of 42ul, incubated at 37deg. C for 20min, 72 deg. C for 5min, and held at 4deg. C. The reaction is then cleaned up using 21ul (0.5X) AMPure XP using two washes with 200ul 80% ethanol, and eluted in 61ul of water. Adapters ligation was set up as follows: 60ul DNA eluate, 25ul Ligation Buffer, 10ul NEBNExt Quick T4 DNA Ligase (NEB E6056S), and 5ul Ligation Adapter, and incubated for 40min at room temperature. The entire ligation reaction was subjected to AMPure XP bead clean up using 40ul (0.4X) of AMPure beads, two washes with Long Fragment Buffer, and eluted in 15ul Elution Buffer. Size distribution and concentration of library was checked by Agilent Tapestation Genomic DNA ScreenTape Analysis. ~75ng-560ng of each library per flow cell was each run on 1 or 2 flow cells on a Promethion P2 Solo, yielding ~100k reads per flow cell run over 40h-72h. Nanopore sequencing of genomic DNA