GSM6754742: ML_scClone5_iPSC_Nanopore; Homo sapiens; OTHER
This protocol was modified from Giesselmann at el (16). Briefly, 1X10e7 hiPSCs were resuspended in 100 μl of 1XPBS. Cells were lysed by adding 10 ml of TLB solution composed of 10 mM Tris-Cl (pH 8), 25 mM EDTA (pH 8), 0.5 % SDS (wt/vol) and 20 μg ml-1 RNase A (Sigma) for 1 h at 37 °C. Then, proteins were digested at 50 °C for 3 hours using 50 μl of proteinase K (BIO-37084). The viscous solution was transferred into a 50-ml Falcon tube containing 5 g of phase-lock gel and 10 ml of ultrapure Phenol/Chloroform/Isoamyl Alcohol (Fisher) was added. Samples were mixed on a rotator at 40 r.p.m. for 10 min and phase separation was performed by centrifugation at 2,800g for 10 min. The aqueous phase was then carefully poured into a fresh 50-ml Falcon tube containing 5 g of phase-lock gel followed by a second phase separation using 10 ml of ultrapure Phenol/Chloroform/Isoamyl Alcohol. Samples were mixed and centrifuged as described above. The aqueous phase was poured into a fresh 50-ml Falcon tube, and the genomic DNA was precipitated using 4 ml of 5 M ammonium acetate together with 30 ml of ice-cold ethanol (100%) and gently inverted ten to twenty times. Precipitated DNA was centrifuged at 12,000g for 5 min and washed with 70% ethanol twice. Supernatant was removed and the DNA pellet was dried at room temperature (RT) for 2–5 min. Rehydration of DNA in 250 μl of 1XTris-EDTA (pH 8) was performed at RT on a rotator for 20 r.p.m. overnight. Samples were stored at 4 °C for 2 days before use. To perform targeted sequencing of FMR1, we designed and synthesized CRISPR–Cas9 crRNAs targeting the genomic regions adjacent to the FMR1 CGG repeats with the ChopChop online tool. The crRNAs used are listed in Supplementary Table 2. Preparation of the Cas9 nucleoprotein complex (Cas9 RNPs) was performed as follows: lyophilized Cas9 crRNA and tracrRNA (IDT) were suspended at 100 μM in TE (pH 7.5). The 4 crRNA probes were pooled for the cleavage reaction by combining equal volumes of each crRNA probe (0.25 μl /each) and 1 μl tracrRNA (100 μM stock) in 8 μl of water. The pooled crRNAs and tracrRNA were annealed with a thermal cycler at 95º C for 5 mins, allowed to cool to room temperature, then spun down to collect any liquid in the bottom of the tube. To form Cas9 RNPs (for 10 reactions), components were assembled in a 1.5 ml Eppendorf DNA LoBind tube in the following order: annealed 10 μl crRNA•tracrRNA pool (10 μM), 10 μl 10x NEB CutSmart buffer, 79.2 μl Nuclease-free water, 0.8 μl HiFi Cas9 (62 μM, IDT). The tube was mixed thoroughly by flicking. RNPs were formed by incubating the tube at room temperature for 30 mins, then returned to ice until required. Meanwhile, dephosphorylated genomic DNA was prepared by assembling the components in a 1.5 ml Eppendorf DNA LoBind tube in the following order: 5 μg of high molecular weight (HMW) DNA in 24 μl, 3 μl NEB CutSmart Buffer (10x) and 3 μl of QuickCIP enzyme (NEBM0525S). The sample was then incubated in a thermocycler at 37º C for 20 minutes, 80º C for 2 minutes, then held at 20º C (room temperature). The reaction was mixed gently by flicking the tube and spun down. 10 μl RNPs from the previous step was incubated with 5 μg of dephosphorylated HMW DNA, 1 μl of 10 mM dATP, and 1 μl of Taq polymerase (NEB) for 60 min at 37 °C on a thermocycler followed by 5 min at 72 °C. 1 μl Proteinase K (Sigma, 20 mg/ml stock concentration) was added to each reaction, and samples were incubated at 43°C for 30 mins to remove proteins for following size selection. The reaction was then purified to remove high concentration salt as follows: Cas9-cut genomic DNA(total volume is 42 ul) was precipitated using 16 ul of 5 M ammonium acetate together with 126 ul of ice-cold ethanol (absolute) and gently inverted ten times. Precipitated DNA was spun down at 16,000g for 5 min. DNA was washed with 500 ul of 70% ethanol and centrifuged at 16,000g for 5 min, and this step was repeated two times. The supernatant was removed and the DNA pellet was dried at RT for 2-5 min. Rehydration of DNA was performed at 50 °C for 1 hour using 200 μl of 10 mM Tris-HCl (pH 8). DNA was further homogenized on a rotator at 37 °C and 20 r.p.m overnight. Size selection was then performed with the Bluepippin BLF7510 (sagescience) using the “0.75DF 3-10kb Marker S1” cassette definition with size range at 5-12 kb.   To perform barcode ligation, the following was performed: 3 μl unique barcode (ONT EXP-NBD104) was added to 50 μl of Blunt/TA Ligase Master Mix (NEB) for each sample. The reactions were incubated at RT for 10 min, spun down, and then put on a magnet. The beads were washed with 200 μl of freshly-prepared 70% ethanol, without disturbing the pellet, twice, and allowed to dry for 30~60 seconds. The remaining pellet was then resuspended in 16 μl nuclease-free water and incubated for 10 minutes at room temperature. The reaction was then placed on a magnet and 16 μl of supernatant was removed into a clean 1.5 ml Eppendorf DNA LoBind tube. Samples were then quantified using a Qubit fluorometer, together with the Qubit dsDNA HS assay kit (Thermo Fisher Scientific).   Adapters were then ligated by first adding 20 μl NEBNext® Quick Ligation Buffer (NEB#E6056S), 10 μl NEBNext Quick T4 DNA ligase(NEB#E6056S), and 5 μl Adapter Mix (AMII) at room temperature in a separate 1.5 ml Eppendorf DNA LoBind Tube. The ligation reaction was mixed thoroughly. 20 μl of the adapter ligation reaction was mixed with the pooled native barcode-ligated samples. Immediately after mixing, the remaining 15 μl of the adapter ligation mix was added to the native barcode-ligated sample, to yield a 100 μl ligation mix. The reaction was incubated for 10 minutes at room temperature. Then 1 volume (100 μl) of TE (pH 8.0) was added to the ligation mix, followed by 0.4x volume (80 μl) of AMPure XP Beads. The sample was then incubated for 10 minutes at room temperature, placed back on the magnet, and the supernatant was removed. The beads were then washed with 250 μl Long Fragment Buffer (LFB) twice and then air-dried for ~30 seconds. The library was eluted off the beads in 14 μl Elution Buffer (EB). 13 uL of the libary was then mixed with 37.5 μl sequencing buffer (SQB) and 25.5 μl loading beads (LB) and loaded onto the MinION flowcell.